Tumor-targeting monoclonal antibodies to FZD10 and uses thereof

ABSTRACT

The present invention relates to an antibody or a fragment thereof which is capable of binding to a Frizzled homologue 10 (FZD10) protein, such as a mouse monoclonal antibody, a chimeric antibody and a humanized antibody. Also, the present invention relates to a method for treating and/or preventing FZD10-associated disease; a method for diagnosis or prognosis of FZD10-associated disease; and a method for in vivo imaging of FZD10 in a subject.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority on U.S. Provisional Application No.60/815,257 filed on Jun. 21, 2006. The entire contents of the aboveapplication are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to an antibody or a fragment thereof whichis capable of binding to a Frizzled homologue 10 (FZD10) protein, suchas a mouse monoclonal antibody, a chimeric antibody and a humanizedantibody. Also, the present invention relates to a method for treatingand/or preventing FZD10-associated disease; a method for diagnosis orprognosis of FZD10-associated disease; and a method for in vivo imagingof FZD10 in a subject.

BACKGROUND OF THE INVENTION

Monoclonal antibodies against cancer-specific molecules have been provedto be useful in cancer treatment (Harris, M. (2004). Lancet Oncol, 5,292-302.). In addition to successful examples of clinical application ofthe humanized or chimeric antibodies such as trastuzumab (Baselga, J.(2004). Oncology, 61, Suupl 2 14-21.), rituximab (Maloney, D. G., et al.(1997). Blood, 90, 2188-95.) and bevacizumab (Ferrara, N., et al.(2004). Nat Rev Drug Discov, 3, 391-400.) for breast cancer, malignantlymphoma and colon cancer, a number of monoclonal antibodies againstother molecular targets are in development and being evaluated theiranti-tumor activities. These monoclonal antibodies are expected toprovide a hope to patients having tumors that have no effectivetreatment. One of the other important issues for these monoclonalantibodies is achievement of selective therapeutic effects to cancercells without severe toxicity due to their specific reaction to cellsexpressing target molecules (Crist, W. M., et al. (2001). J Clin Oncol,19, 3091-102; Wunder, J. S., et al. (1998). J Bone Joint Surg Am, 80,1020-33; Ferguson, W. S. and Goorin, A. M. (2001). Cancer Invest, 19,292-315.).

Among soft tissue sarcomas, osteosarcoma, Ewing's sarcoma andrhabdomyosarcoma are sensitive to chemotherapy and these diseases can bewell managed by chemotherapy. On the other hand, spindle cell sarcomasare resistant to chemo- and radiotherapy and patients with them usuallyexhibit poor prognosis. For synovial sarcoma (SS), surgical treatment iseffective for patients at an early stage, but no effective therapeuticdrug is available to those at an advanced stage. Hence, development ofnovel therapeutic modalities is expected to improve patients' prognosisbetter.

Genome-wide gene expression analysis in tumors provides the usefulinformation to identify the new molecular targets for development ofnovel anticancer drugs and tumor markers. In previous study, the presentinventors have analyzed gene-expression profile of several soft tissuesarcomas using genome-wide cDNA microarray consisting of 23,040 genesand demonstrated that Frizzled homologue 10 (FZD10) (GenBank AccessionNOs. AB027464 (SEQ ID NO:1) and BAA84093 (SEQ ID NO:2)) was up-regulatedspecifically and frequently in SSs (Nagayama, S., et al. (2002) CancerRes, 62, 5859-66; and WO2004/020668). FZD10 gene product is a member ofFrizzled family and a putative WNT signal receptor (Koike, J., et al.(1999). Biochem Biophys Res Commun, 262, 39-43.). Further analysisshowed that FZD10 is expressed specifically in SS, and at no orhardly-detectable level in other normal organs except the placenta,suggesting that therapeutics targeting this molecule would cause no orlittle adverse reaction (Nagayama, S., et al. (2002). Cancer Res, 62,5859-66.). RNAi experiments implicated that FZD10 was significantlyinvolved in the tumor growth of SS (WO2006/013733). Furthermore, thepresent inventors generated the rabbit polyclonal antibody against theextracellular domain of FZD10 (FZD10-ECD), and found that this antibodyhad antitumor activity in mouse xenograft model of SS (Nagayama, S., etal. (2005). Oncogene, 24, 6201-12; and WO2005/004912). Together, theantibody therapy against FZD10 could be expected to improve the clinicaloutcome of SS.

SUMMARY OF THE INVENTION

Hereinbelow, it is reported that generation of the murine monoclonalantibodies against FZD10 by means of cell-immunization method forpossible clinical application. In vivo tumor-binding activity of theseantibodies was evaluated using fluorescent in vivo imaging system withnear-infrared fluorescence in addition to the conventional method withradionuclides. Here, we reveal the binding specificity of anti-FZD10monoclonal antibodies both in vitro and in vivo as well asinternalization of these antibodies in the cells expressing FZD10, andfound that SYO-1-bearing xenograft mice treated with a single tail vainof ⁹⁰Y-labeled anti-FZD10 Mab at 100 μCi dose was observed significantantitumor effect.

Based on the above findings, the present inventors concluded that themurine monoclonal antibodies against FZD10 have therapeutic potential inthe treatment and diagnosis of SS and other FZD10-overexpressing tumors.

Therefore, in the first aspect, the present invention provides anantibody or a fragment thereof, which comprises an H (heavy) chain V(variable) region comprising a complementarity determining region (CDR)having the amino acid sequences shown in SEQ ID NOs: 15, 17 and 19 or aCDR functionally equivalent thereto and an L (light) chain V regioncomprising a CDR having the amino acid sequences shown in SEQ ID NOs:23, 25 and 27 a CDR functionally equivalent thereto, and which iscapable of binding to a Frizzled homologue 10 (FZD10) protein or apartial peptide thereof.

In one embodiment, the antibody or fragment thereof is selected from thegroup consisting of a mouse antibody, a chimeric antibody, a humanizedantibody, an antibody fragment, and single-chain antibody.

In a preferred embodiment, the antibody is a mouse antibody. Preferably,the mouse antibody comprises an H chain having the amino acid sequenceshown in SEQ ID NO: 57 and/or an L chain having the amino acid sequenceshown in SEQ ID NO: 59. For example, the mouse antibody can be producedby the hybridoma clone 92-13 (FERM BP-10628).

In an alternative preferred embodiment, the antibody is a chimericantibody. Preferably, the chimeric antibody comprises an H chain Vregion having the amino acid sequence shown in SEQ ID NO: 13, forexample, the chimeric antibody may comprise an H chain having the aminoacid sequence shown in SEQ ID NO: 46. Preferably, the chimeric antibodycomprises an L chain V region having the amino acid sequence shown inSEQ ID NO: 21, for example, the chimeric antibody may comprise an Lchain having the amino acid sequence shown in SEQ ID NO: 48.

More preferably, the chimeric antibody comprises an H chain V regionhaving the amino acid sequence shown in SEQ ID NO: 13 and an L chain Vregion having the amino acid sequence shown in SEQ ID NO: 21. Forexample, the chimeric antibody comprises an H chain having the aminoacid sequence shown in SEQ ID NO: 46 and an L chain having the aminoacid sequence shown in SEQ ID NO: 48.

In one embodiment, the chimeric antibody further comprises a humanantibody C (constant) region.

In an alternative preferred embodiment, the antibody is a humanizedantibody. In one embodiment, the humanized antibody further comprises ahuman antibody FR (framework) region and/or a human antibody C region.

In the second aspect, the present invention provides an antibody or afragment thereof, which comprises an H (heavy) chain V (variable) regioncomprising a complementarity determining region (CDR) having the aminoacid sequences shown in SEQ ID NOs: 31, 33 and 35 or a CDR functionallyequivalent thereto and an L (light) chain V region comprising a CDRhaving the amino acid sequences shown in SEQ ID NOs: 39, 41 and 43 or aCDR functionally equivalent thereto, and which is capable of binding toa Frizzled homologue 10 (FZD10) protein or a partial peptide thereof.

In one embodiment, the antibody or fragment thereof is selected from thegroup consisting of a mouse antibody, a chimeric antibody, a humanizedantibody, an antibody fragment, and single-chain antibody.

In a preferred embodiment, the antibody is a mouse antibody. Preferably,the mouse antibody comprises an H chain having the amino acid sequenceshown in SEQ ID NO: 61 and/or an L chain having the amino acid sequenceshown in SEQ ID NO: 63. For example, the mouse antibody can be producedby the hybridoma clone 93-22 (FERM BP-10620).

In an alternative preferred embodiment, the antibody is a chimericantibody. Preferably, the chimeric antibody comprises an H chain Vregion having the amino acid sequence shown in SEQ ID NO: 29, forexample, the chimeric antibody comprises an H chain having the aminoacid sequence shown in SEQ ID NO: 50. Preferably, the chimeric antibodycomprises an L chain V region having the amino acid sequence shown inSEQ ID NO: 37, for example, the chimeric antibody comprises an L chainhaving the amino acid sequence shown in SEQ ID NO: 52.

More preferably, the chimeric antibody comprises an H chain V regionhaving the amino acid sequence shown in SEQ ID NO: 29 and an L chain Vregion having the amino acid sequence shown in SEQ ID NO: 37. Forexample, the chimeric antibody comprises an H chain having the aminoacid sequence shown in SEQ ID NO: 50 and an L chain having the aminoacid sequence shown in SEQ ID NO: 52.

In one embodiment, the chimeric antibody further comprises a humanantibody C (constant) region.

In an alternative preferred embodiment, the antibody is a humanizedantibody. In one embodiment, the humanized antibody further comprises ahuman antibody FR (framework) region and/or a human antibody C region.

In yet an alternative embodiment, the antibody or fragment thereof canbe labeled with a radioisotope label or a fluorescent label. Suchradioisotope label includes ⁹⁰yttrium (⁹⁰Y), ¹²⁵iodine (¹²⁵I) and¹¹¹indium (¹¹¹In).

In the third aspect, the present invention provides a hybridoma clone92-13 (FERM BP-10628) which produces the mouse monoclonal antibody92-13.

In the forth aspect, the present invention provides a hybridoma clone93-22 (FERM BP-10620) which produces the mouse monoclonal antibody93-22.

In the fifth aspect, the present invention provides a method fortreating or preventing a disease that is associated with Frizzledhomologue 10 (FZD10) in a subject, comprising administering to thesubject an effective amount of the antibody or fragment above. In oneembodiment, the disease that is associated with FZD10 is selected fromsynovial sarcoma (SS), colorectal cancer, gastric cancer, chronicmyeloid leukemia (CML), and acute myeloid leukemia (AML).

In the sixth aspect, the present invention provides a method fordiagnosis or prognosis of a disease that is associated with Frizzledhomologue 10 (FZD10) or of a predisposition to develop the disease in asubject, comprising

(a) contacting a sample or a specimen from the subject with the antibodyor fragment above;

(b) detecting the FZD10 protein in the sample or specimen; and

(c) judging whether or not the subject suffers from or is at risk ofdeveloping the disease based on the relative abundance of the FZD10protein compared to a control.

In one embodiment, the disease that is associated with FZD10 is selectedfrom synovial sarcoma (SS), colorectal cancer, gastric cancer, chronicmyeloid leukemia (CML), and acute myeloid leukemia (AML).

In the seventh aspect, the present invention provides a method for invivo imaging of Frizzled homologue 10 (FZD10) protein in a subject,comprising administering to the subject an effective amount of theantibody or fragment above.

In the eighth aspect, the present invention provides a pharmaceuticalcomposition for treating or preventing a disease associated withFrizzled homologue 10 (FZD10), comprising the antibody or fragment aboveand a pharmaceutically acceptable carrier or excipient.

In the ninth aspect, the present invention provides a kit for diagnosisor prognosis of a disease associated with Frizzled homologue 10 (FZD10),comprising the antibody or fragment above.

In the tenth aspect, the present invention provides a pharmaceuticalcomposition for in vivo imaging of Frizzled homologue 10 (FZD10)protein, comprising the antibody or fragment above.

In the eleventh aspect, the present invention provides use of theantibody or fragment above in the manufacture of a kit for diagnosis orprognosis of a disease associated with Frizzled homologue 10 (FZD10).

In the twelfth aspect, the present invention provides use of theantibody or fragment above in the manufacture of a composition forprevention or treatment of a disease associated with Frizzled homologue10 (FZD10).

The term “disease that is associated with FZD10” (FZD10-associateddisease) refers to a disease that is associated with over-expression ofFZD10 protein. Such diseases include, but are not limited to, synovialsarcoma (SS), colorectal cancer, gastric cancer, chronic myeloidleukemia (CML), and acute myeloid leukemia (AML).

The term “fragment” means any antibody fragment that can be preparedfrom the antibody against FZD10 protein and contains defined CDRs. Suchfragment includes, but not limited to, Fab fragment, F (ab′)₂ fragment,and Fv fragment.

The term “modified antibody” means any antibody that can be derived fromthe antibody against FZD10 and contains defined CDRs. Such modifiedantibody includes, but not limited to, a PEG-modified antibody. Theantibody fragment or modified fragment can be readily recognized by aperson skilled in the art and produced by using any methods known in theart.

The term “subject” herein refers to a subject who has suffered fromFZD10-associated disease and also a subject suspected to haveFZD10-associated disease. The subject in the present invention may beanimals including mammals and avian animals. For example, mammals mayinclude humans, mice, rats, monkeys, rabbits, and dogs.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the office upon request and paymentof the necessary fee.

FIGS. 1 a to 1 f show characterization of binding specificity for twoanti-FZD10 monoclonal antibodies.

FIG. 1 a shows flow-cytometric analysis of the four antibodies, 39-2 and39-10 (disclosed in WO2005/004912), 92-13 and 93-22, using five SS lines(SYO-1, YaFuSS, HS-SY-2, Fuji and 1973/99) and one colon-cancer cellline (LoVo). Solid lines show the fluorescent intensity detected by eachmAbs; broken lines depict the fluorescent intensities of cells incubatedwith non-immunized mouse IgG as a negative control.

FIG. 1 b shows semi-quantitative RT-PCR of FZD10 in the same tumor-celllines as used in FIG. 1 a. Expression of β2-microglobulin gene (β2MG)served as an internal control.

FIG. 1 c shows flow-cytometric analysis of 92-13 (top panels) and 93-22(lower panels) against exogenous FZD10 were indicated. Colon cancer cellline, SNU-C5 was transfected with pCAGGS empty vector (left panels) orpCAGGS-FZD10-myc/His (right panels) and analyzed 48 hours aftertransfection. Solid lines show the fluorescent intensity detected byeach mAbs; broken lines depict the fluorescent intensities of cellsincubated with non-immunized mouse IgG as a negative control.

FIG. 1 d shows binding of ¹²⁵I-labeled 39-10, 39-2, 92-13 and 93-22 tonormal human blood cells. Radio-labeled Mabs were incubated with eachnormal human fresh blood of three individuals (A, B and C) in theabsence (open bar) or presence (closed bar) of non-labeled identicalantibodies.

FIG. 1 e shows binding activity of ¹²⁵I-labeled Mabs. A constant amountof radio-labeled Mabs was incubated with SYO-1 cell and increasingamount of non-labeled antibodies. The percent radioactivity bound tocells was plotted against the amount of non-labeled antibody. Closedcircle; 92-13, Open circle; 93-22.

FIG. 1 f shows flow-cytometric analysis of self-block and cross-block.Alexa-488-labeled 92-13 (Top panels) and 93-22 (Lower panels) wereincubated with SYO-1 cell in (i) PBS, or in the presence of 100 μg of(ii) non-labeled 92-13 and (iii) non-labeled 93-22. Shaded histogramshow the fluorescent intensity detected by each Alexa488-labeled Mabs;broken lines depict the fluorescent intensities of cells incubated withPBS as a negative control.

FIG. 2 shows immunohistochemical analyses in SS and normal human frozentissue sections with no antibody (a, d, g, j, and m), 92-13 (b, e, h, k,and n) and 93-22 (c, f, i, l, and o). (a-c), synovial sarcoma; (d-f),kidney; (g-i), liver, (j-l), heart; (m-o), brain. Originalmagnification: ×100.

FIG. 3 shows biodistribution of ¹¹¹In-labeled and ¹²⁵I-labeledantibodies. 10 kBq of (a), ¹¹¹In-labeled 92-13, (b), ¹²⁵I-labeled 92-13,(c), In-labeled 93-22 and (d), ¹²⁵I-labeled 93-22 were injectedintravenously into SYO-1 tumor bearing BALB/c nude mice. The organs andtumor were dissected at one hour (open bar), 24 hours (hatched bar) and48 hours (closed bar), and the radioactivities were measured. The datashown is the representative data in two independent experiments.

FIG. 4 a shows in vivo fluorescence imaging of SYO-1 tumor-bearing miceafter injection of Alexa 647-labeled 92-13 or 93-22.Fluorescence-labeled Mabs were administered at a dose of 20 μg per mouseintraperitoneally. All fluorescence images were acquired with a60-second exposure time (f/stop=2) before injection, immediately afterinjection (0 hour), 24, 48 and 96 hours. The arrows indicate theposition of the tumor. S.C. tumor is located in dorsal for 92-13 (toppanels) and in trunk for 93-22 (lower panels). Fluorescence signal fromAlexa647 was pseudo-colored according to the color bar indicated onright. In 93-22 (lower panel), the arrowheads indicate the position ofinjection.

FIGS. 4 b and 4 c show representative images of dissected organs andtumors from mice shown in FIGS. 4 a, 4 b; 92-13, and 4 c; 93-22. i,SYO-1 tumor; ii, liver; iii, spleen; iv, kidney; v, pancreas; vi, colon.

FIG. 5 a shows in vivo fluorescence imaging of LoVo tumor-bearing miceafter injection of Alexa647-labeled 92-13 or 93-22. Fluorescence-labeledMabs were administered as FIG. 4. All fluorescence images were acquiredwith a 60-second exposure time (f/stop=2) immediately after injection (0hour), 48, 72, 96 and 120 hours (h). Arrow indicates the position of thetumor. S.c. tumor is located in right forearm both for 92-13 (Toppanels) and 93-22 (lower panels).

FIGS. 5 b and 5 c show representative images of dissected organs andtumors of mice shown in FIGS. 5 a. 5 b; 92-13 and 5 c; 93-22. i, LoVotumor; ii, liver; iii, spleen; iv, kidney; v, pancreas; vi, colon.

FIG. 6 shows internalization of 92-13 and 93-22 was assessed by confocalmicroscopy. Cells were treated with PBS (a, d, and g), 50 μg/ml of 92-13(b, e, and h) or 93-22 (c, f, i) for 3 hours in 37° C., 5% CO₂.Antibodies bound to the cell surface were acid-stripped with 0.1Mglycine buffer (pH2.5). Cells were fixed, permeabilized and then blockedwith 3% BSA. Intracellular antibodies were detected with goat anti-mouseIgG-Alexa488 and nucleus was stained with DAPI. (a-c), SYO-1; (d-f),YaFuSS; (g-i) Lovo.

FIG. 7 shows the effect of ⁹⁰Y-labeled 92-13 on tumor growth. Whentumors were established (0.4-2.7 cm³), mice were given a single tailvain of 100 μCi of ⁹⁰Y-labeled 92-13.

FIG. 8 shows both chimeric 92-13 and 93-22 induced ADCC specifically tothe FZD10-overexpressing SYO-1 cells. 1 μg/ml of chimeric 93-22 antibody(ch93-22) or chimeric 92-13 antibody (ch92-13) at variousEffector:Target ratio. PBMC from various donors were used as Effectorcell; (a), (c) ADCC of chimeric 92-13 against SYO-1 cell with fivehealthy human PBMC donors. (b), (d) ADCC of chimeric 93-22 against LoVocell with two healthy human PBMC donors. Quantification of cytotoxitywith LDH activity is described in (Nagayama, S., et al. Oncogene, 24,6201-12.).

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS OF THE PRESENT INVENTION

Frizzled homologue 10 (FZD10) is a member of Frizzled family, which is areceptor of Wnt signaling. As described hereinbelow, we successfullyestablished murine monoclonal antibodies and chimeric antibodies againstFZD10 protein that may be useful for medical use.

The murine monoclonal FZD10-specific antibodies (92-13 and 93-22 Mabs)are established by immunizing mice with FZD10-transfected cells. Both92-13 and 93-22 Mabs were shown to have specific binding activityagainst FZD10 in SS cell line, SYO-1 cells and FZD10-transfected COS7cells by using flow cytometry (FACS) analysis. To validate the specificbinding activity of those antibodies in vivo, the present inventorsinjected fluorescent-labeled Mabs intraperitoneally or intravenouslyinto the mice carrying SS xenografts and found that these Mabs werebound to the FZD10-expressing tumors, but not to any other normal mousetissues by the use of the in vivo fluorescent imaging system andradioactivities. Subsequent immunohistochemical analyses with the Mabsconfirmed an absence or hardly-detectable level of FZD10 protein innormal human organs except the placenta. Furthermore, interestingly thepresent inventors found that the Mabs were internalized into the SS cellline, SYO-1, but not into FZD10-negative cell line, LoVo using confocallaser scanning microscopy. Surprisingly, SYO-1-bearing xenograft micetreated with a single tail vain of ⁹⁰Y-labeled anti-FZD10 (92-13) Mab at100 μCi dose was observed significant antitumor effect. Taken together,we conclude that these specific Mabs against FZD10 could be utilized asthe novel diagnostic marker or treatment of SS with minimal or no riskof adverse reactions.

Due to their complicated protein structure, it is often very difficultto generate antibodies against seven-transmembrane proteins. In previousstudy, the present inventors demonstrated that FZD10 formedhomo-oligomer (Nagayama, S., et al. (2005). Oncogene, 24, 6201-12.).After failure of multiple attempts to generate anti-FZD10 monoclonalantibodies that could recognize a native form of FZD10 by the use offull-length or partial recombinant FZD10 proteins, we finally applied toimmunization by injection of living COS-7 cells overexpressing FZD10into the foot-pad of Balb/c-mice and successfully obtained the twohybridomas producing anti-FZD10 antibodies that had an ability torecognize the native FZD10 form in living cells by FACS analysis. Sincethose antibodies did not detect FZD10 protein in western blotting, thepresent inventors assume that those Mabs recognize the tertiarystructure of FZD10.

To investigate the in vivo distribution of 92-13 and 93-22 Mabs, thepresent inventors applied two methods; one based on the radionuclidemodalities using ¹²⁵I and ¹¹¹In-labeled antibodies, and the other basedon the fluorescence imaging using near-infrared-labeled (Alexa647)antibodies. Near-infrared fluorescent, mostly indocyanine dye, is nowwidely used in the in vivo imaging for diagnostic purpose because thelight of this wavelength penetrates living tissue quite efficiently(Chen, X., et al. (2004). Cancer Res, 64, 8009-14.). The resultsobtained two approaches were very concordant and indicated that 92-13and 93-22 bound to SYO-1 tumor cells, but not to other normal tissues.To confirm whether those antibodies can be applied for clinical use, thepresent inventors further examined the binding activity of antibodiesagainst normal blood cells. The binding activity of ¹²⁵I-labeled 92-13and 93-22 against normal human blood cells were undetectable in all ofthree individual donors (FIG. 1 d). These results were consistent withthose of FACS analysis using human peripheral blood mononuclear cell(data not shown), suggesting clinical applicability of these twoantibodies with little possibility of adverse effect to SS patientsbecause of very specific binding affinity to the FZD10 molecule.Furthermore, in vitro experiments using confocal microscopy reveled thatthe specific binding of 92-13 and 93-22 Mabs to cell-surface FZD10induced the internalization of the antibodies (FIG. 6). As describedpreviously (Stein, R., et al. (2001). Criti Rev Oncol Hematol, 39,173-80; Stein, R., et al. (2005). Clin Cancer Res, 11, 2727-34.), iflabeled Mabs is internalized after binding, ¹²⁵I-labeled antibody ismetabolized in the lysosomes and diffused from target tumor cells where¹¹¹In-labeled antibody remains in the lysosomes. As observed in FIG. 3,the radioactivities of ¹¹¹In-labeled antibody and ¹²⁵I-labeled antibodyin tumors were significantly different (FIG. 3, a and b, c and d). Thesefindings suggest that 92-13 (and 93-22) Mabs can specificallyinternalize into the SS cells via FZD10 protein.

When antibodies are applied to cancer therapy, the following threemechanisms are thought to exert the anti-tumor activity; (i) in casethat the target molecule is involved in growth enhancement,neutralization of antibodies would block the growth signal transductionand then suppress the growth of tumor cells; (ii) The second possibilityis the effecter activities to induce antibody-dependent cell-mediatedcytotoxity (ADCC) or complement-dependent cytotoxity (CDC). (iii) Thethird case is radionuclides or antitumor drug that is conjugated toantibodies and is delivered to the target tumor cells effectively.Although the present inventors previously demonstrated that the targetmolecule FZD10 is involved in the SS tumor growth, neither Mabs 92-13nor 93-22 showed the neutralizing effect in vitro when added to the cellculture media (data not shown) or in vivo when injected to thetumor-bearing mice (data not shown).

Conjugating radionuclide or anti-cancer drug to antibodies such asZevalin (anti-CD20 antibody conjugated with ⁹⁰yttrium) and Mylotarg.(anti-CD33 antibody conjugated with calicheamicin), has been proven tobe highly effective to confer cytotoxity to the antibodies (Wiseman, G.A. and Witzig, T. E. (2005). Cancer Biother Radiopharm, 20, 185-8; vander Velden, V. H., et al. (2001). Blood, 97, 3197-204; Carter, P.(2001). Nat Rev Cancer, 1, 118-29.). Mylotarg exerts its antitumoractivity by releasing antitumor drug, calicheamicin within the cancercell after it was internalized (van der Velden, V. H., et al. (2001).Blood, 97, 3197-204.). In the Examples, for therapeutic experiments,⁹⁰yttrium-DTPA-92-13 conjugate was generated and its antitumor activitywas investigated. In mouse xenograft model, tumors quickly diminishedafter treatment of ⁹⁰yttrium-DTPA-92-13 (FIG. 7). Noticeably, the tumorsincluding larger volume (>1 cm³) of tumor showed no refraction until 34days after administration and no strong toxicity was observed. Sinceanti-FZD10 antibodies 92-13 and 93-22 were likely to be effectivelyinternalized into antigen-positive cells as shown in FIG. 6, conjugationof anti-cancer drug to both Mabs 92-13 and 93-22 is also expected toexert the high anti-cancer effect to SS cells. Referring to the effectoractivity, both chimeric 92-13 and 93-22 induced ADCC specifically to theFZD10-overexpressing SYO-1 cells (FIG. 8, a and c), but not to theFZD10-negative LoVo cells (FIG. 8, b and d). Particularly, chimeric92-13 showed higher induction of cytotoxity as compared with chimeric93-22, however, their activity depends on effector cell donor, possiblycaused by polymorphism of Fc receptor. In conclusion, the presentinventors successfully produced monoclonal antibodies which were able tobind specific to FZD10 on FZD10-overexpressing tumor cells in vitro andin vivo. Together, the present inventors are confident that anti-FZD10monoclonal antibodies have great potential for development of novel drugtherapies for treatment of SS and other tumors that over-express FZD10.

1. Production of an Antibody

Antibodies that can be used in the present invention specifically reactagainst an FZD10 protein derived from an FZD10-associated disease. Theterm “antibody” used herein means an antibody molecule as a whole, orits fragments such as Fab fragments, F(ab′)₂ fragments and Fv fragments,which can bind to the protein or its partial peptides as the antigen.The antibody can be either a polyclonal antibody or a monoclonalantibody. It can also be a humanized or chimeric antibody, or a singlechain Fv (scFv) antibody. The antibodies (polyclonal antibodies andmonoclonal antibodies) for use in the present invention can be prepared,for example, by the following process.

(1) Monoclonal Antibody

Initially, an antigen is prepared for the production of an antibodyuseful in the present invention. FZD10 protein or its partial peptidecan be used as an immunogenic protein. Alternatively, a cell expressingFZD10 protein or its partial peptide can also be used as an immunogen.The amino acid sequence of FZD10 protein used as the immunogen in thepresent invention and the cDNA sequence encoding the protein arepublicly available in GenBank as Accession Nos. BAA84093 (SEQ ID NO: 1)and AB027464 (SEQ ID NO: 2), respectively. The FZD10 protein or itspartial peptide for use as the immunogen can be synthetically preparedaccording to a procedure known in the art such as a solid-phase peptidesynthesis process, using the available amino acid sequence information.The partial peptides of FZD10 protein include, but are not limited to, apeptide containing residues 1-225 of the amino acid sequence shown inSEQ ID NO: 1, which corresponds to the N-terminal extracellular domainof FZD10 protein (FZD10-ECD).

The protein or its partial peptide, or the cell expressing them can beprepared by using the sequence information of cDNA encoding FZD10protein or its partial peptide according to a known gene recombinationprocedure. The production of the protein or its partial peptide as wellas the cell expressing them according to such a gene recombinationprocedure will be illustrated below.

A recombinant vector for the production of protein can be obtained bylinking the above cDNA sequence to an appropriate vector. A transformantcan be obtained by introducing the recombinant vector for the productionof protein into a host so that the target FZD10 protein or its partialpeptide can be expressed.

As the vector, a phage or plasmid that is capable of autonomouslyreplicating in a host is used. Examples of a plasmid DNA include pCAGGS,pET28, pGEX4T, pUC118, pUC119, pUC18, pUC19, and other plasmid DNAsderived from Escherichia coli; pUB110, pTP5, and other plasmid DNAsderived from Bacillus subtilis; and YEp13, YEp24, YCp50 and otherplasmid DNAs derived from yeast. Examples of a phage DNA include lambdaphages such as λgt11 and λZAP. In addition, animal virus vectors such asretrovirus vector and vaccinia virus vector can be used, and insectvirus vectors such as baculovirus vector can also be used.

The DNA encoding the FZD10 protein or its partial peptide (hereinafterreferred to as FZD10 DNA) is inserted into the vector, for example, bythe following method. In this method, purified DNA is cleaved by anappropriate restriction enzyme and inserted into a restriction enzymesite or a multi-cloning site of an appropriate vector DNA to ligate intothe vector.

In addition to a promoter and the FZD10 DNA, any of enhancers and othercis elements, splicing signals, poly A addition signals, selectivemarkers, ribosome binding site (RBS), and other elements can be ligatedinto the recombinant vector for the production of protein for use inmammalian cells, if desired.

For ligating the DNA fragment to the vector fragment, a known DNA ligasecan be used. The DNA fragment and the vector fragment are annealed andligated, thereby producing a recombinant vector for the production of aprotein.

The host for use in transformation is not specifically limited as longas it allows the FZD10 protein or its partial peptide to be expressedtherein. Examples of the host include bacteria, for example, E. coli,and Bacillus; yeast, for example, Saccharomyces cerevisiae; animalcells, for example, COS cells, Chinese Hamster Ovary (CHO) cells, andinsect cells.

For example, when a bacterium is used as the host, the recombinantvector for the protein production should preferably be capable ofautonomously replicating in the host bacterium and comprise a promoter,a ribosome binding site, the FZD10 DNA, and a transcription terminationsequence. The recombinant vector may further comprise a gene forregulating the promoter. An example of Escherichia coli includesEscherichia coli BRL, and an example of Bacillus is Bacillus subtilis.Any promoter that can be expressed in the host such as Escherichia colican be used herein.

The recombinant vector can be introduced into the host bacterium by anyprocedures known in the art. Such procedures include, for example, amethod using calcium ions and an electroporation. When yeast cell, ananimal cell, or an insect cell is used as the host, a transformant canbe produced according to a known procedure in the art, and then theFZD10 protein or its partial peptide can be produced in the host(transformant).

The FZD10 protein or its partial peptide for use as the immunogen in thepresent invention can be obtained from a culture of the above-generatedtransformant. The “culture” refers to any of culture supernatant,cultured cells, cultured microorganisms, and homogenates thereof. Thetransformant is cultured in a culture medium by a conventional processof culturing a host.

The culture medium for culturing the transformant obtained by usingEscherichia coli, yeast, or other microorganisms as the host can beeither a natural medium or a synthetic medium, as long as it comprises acarbon source, nitrogen source, inorganic salts, and other componentsutilizable by the microorganism and enables the transformant to growefficiently.

The transformant is generally cultured by shaking culture or aerationculture with stirring under aerobic conditions at 25° C. to 37° C. for 3to 6 hours. During culturing, pH is held at a level near neutrality byadjustment with, for example, an inorganic or organic acid, and analkaline solution. During culturing, antibiotics such as ampicillin ortetracycline may be added to the medium according to the selectivemarker inserted into the recombinant expression vector, if necessary.

After culturing, when the FZD10 protein or its partial peptide isproduced within the microorganism or cell, the protein or its partialpeptide is extracted by homogenizing the microorganism or cell. When theFZD10 protein or its partial peptide is secreted from the microorganismor cell, the culture medium is used as is, or debris of themicroorganism or cell is removed from the culture medium, for example,by centrifugation. Thereafter, the FZD10 protein or its partial peptidecan be isolated from the culture and purified by a conventionalbiochemical method for the isolation and purification of proteins, suchas ammonium sulfate precipitation, gel chromatography, ion-exchangechromatography, and affinity chromatography, either individually or incombination.

Whether or not the FZD10 protein or its partial peptide has beenobtained can be confirmed, for example, by SDS polyacrylamide gelelectrophoresis.

Next, the obtained FZD10 protein or its partial peptide, or thetransformant is dissolved in a buffer to prepare an immunogen. Wherenecessary, an adjuvant can be added thereto for effective immunization.Such adjuvants include, for example, commercially available Freund'scomplete adjuvant and Freund's incomplete adjuvant. Any of theseadjuvants can be used alone or in combination.

The immunogen so prepared is administered to a mammal such as a rabbit,rat, or mouse. The immunization is performed mainly by intravenous,subcutaneous, or intraperitoneal injection. The interval of immunizationis not specifically limited and the mammal is immunized one to 3 timesat intervals ranging from several days to weeks. Antibody-producingcells are collected 1 to 7 days after the last immunization. Examples ofthe antibody-producing cells include spleen cells, lymph node cells, andperipheral blood cells.

To obtain a hybridoma, an antibody-producing cell and a myeloma cell arefused. As the myeloma cell to be fused with the antibody-producing cell,a generally available established cell line can be used. Preferably, thecell line used should have drug selectivity and properties such that itcan not survive in a HAT selective medium (containing hypoxanthine,aminopterin, and thymidine) in unfused form and can survive only whenfused with an antibody-producing cell. Possible myeloma cells include,for example, mouse myeloma cell lines such as P3X63-Ag.8.U1 (P3U1), andNS-I.

Next, the myeloma cell and the antibody-producing cell are fused. Forthe fusion, these cells are mixed, preferably at the ratio of theantibody-producing cell to the myeloma cell of 5:1, in a culture mediumfor animal cells which does not contain serum, such as DMEM andRPMI-1640 media, and fused in the presence of a cell fusion-promotingagent such as polyethylene glycol (PEG). The cell fusion may also becarried out by using a commercially available cell-fusing device usingelectroporation.

Then, the hybridoma is picked up from the cells after above fusiontreatment. For example, a cell suspension is appropriately diluted with,for example, the RPMI-1640 medium containing fetal bovine serum and thenplated onto a microtiter plate. A selective medium is added to eachwell, and the cells are cultured with appropriately replacing theselective medium. As a result, the cells that grow about 30 days afterthe start of culturing in the selective medium can be obtained as thehybridoma.

The culture supernatant of the growing hybridoma is then screened forthe presence of an antibody that reacts with the FZD10 protein or itspartial peptide. The screening of hybridoma can be performed accordingto a conventional procedure, for example, using enzyme-linkedimmunosorbent assay (ELISA), enzyme immunoassay (EIA) orradioimmunoassay (RIA). The fused cells are cloned by the limitingdilution to establish a hybridoma, which produces the monoclonalantibody of interest.

The monoclonal antibody can be collected from the established hybridoma,for example, by a conventional cell culture method or by producing theascites. If necessary, the antibody can be purified in theabove-described antibody collecting method according to a knownprocedure such as ammonium sulfate precipitation, ion-exchangechromatography, gel filtration, affinity chromatography, or acombination thereof.

The globulin type of the monoclonal antibodies useful in the presentinvention is not specifically limited, as long as they are capable ofspecifically binding to the FZD10 protein and can be any of IgG, IgM,IgA, IgE, and IgD. Among them, IgG and IgM are preferred.

In the present invention, murine monoclonal antibodies 93-22 and 92-13are successfully established and preferably used. The hybridoma clone93-22 producing mouse monoclonal antibody 93-22 was deposited by ShuichiNakatsuru internationally at the IPOD International Patent OrganismDepository of the National Institute of Advanced Industrial Science andTechnology (AIST Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi,Ibaraki-Ken, 305-8566 Japan) as of Jun. 14, 2006 under the depositnumber of FERM BP-10620. Also, hybridoma clone 92-13 producing mousemonoclonal antibody 92-13 was deposited by Shuichi Nakatsuruinternationally at the IPOD International Patent Organism Depository ofthe National Institute of AIST as of Jun. 28, 2006 under the depositnumber of FERM BP-10628. The monoclonal antibody produced by thehybridoma may be preferably used in the present invention.

In the present invention, a recombinant-type monoclonal antibody mayalso be used, which can be produced by cloning an antibody gene from thehybridoma, integrating the antibody gene into a suitable vector,introducing the vector into a host, and producing the antibody from thehost according to a conventional genetic recombination technique (see,for example, Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192,767-75).

More specifically, mRNA encoding variable (V) region of the anti-FZD10mouse monoclonal antibody is isolated from the antibody-producinghybridoma (for example, those described above). The isolation of themRNA is performed by preparing a total RNA by any known method, such asguanidium ultracentrifugation method (Chirgwin, J. M. et al.,Biochemistry (1979) 18, 5294-9) and AGPC method (Chomczynski, P. et al.,Anal. Biochem. (1987) 162, 156-9), and then producing the desired mRNAfrom the total RNA using mRNA Purification Kit (Pharmacia) or the like.Alternatively, the mRNA may also be prepared directly using QuickPrepmRNA Purification Kit (Pharmacia).

Next, cDNA for the antibody V-region is synthesized from the mRNA with areverse transcriptase. The synthesis of the cDNA may be performed usinga commercially available kit, for example, Gene Racer™ Kit (lnvitrogen).The cDNA may also be synthesized or amplified by 5′-RACE method(Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002;Belyaysky, A. et al., Nucleic Acids Res. (1989) 17, 2919-32) using5′-Ampli FINDER RACE Kit (Clontech) in combination with a PCR method.

The amino acid sequences of H chain and L chain of mouse monoclonalantibody 92-13 are shown in SEQ ID NO: 57 and 59, respectively (encodedby the nucleotide sequence as shown in SEQ ID NO: 58 and 60,respectively). The amino acid sequences of H chain and L chain of mousemonoclonal antibody 93-22 are shown in SEQ ID NO: 61 and 63,respectively (encoded by the nucleotide sequence as shown in SEQ ID NO:62 and 64, respectively). Based on the sequence information, primersused for amplifying the H chain or L chain of mouse monoclonal antibodyof interest can be designed using a conventional method.

A DNA fragment of interest is isolated and purified from the resultantPCR product and then ligated to a vector DNA to obtain a recombinantvector. The recombinant vector is introduced into a host such as E.coli, and a colony containing a desired recombinant vector is selected.The nucleotide sequence of the DNA of interest in the recombinant vectoris confirmed using, for example, an automated sequencer.

Once DNA encoding the anti-FZD10 antibody V-region is obtained, the DNAis integrated into an expression vector containing DNA encoding theantibody constant (C) region.

For the production of the anti-FZD10 antibody used in the presentinvention, the antibody gene is integrated into an expression vector sothat the antibody gene can be expressed under the control of expressioncontrol elements (e.g., enhancer, promoter). A host cell is transformedwith the expression vector to express the antibody.

In the expression of the antibody gene, DNA encoding heavy (H) chain andDNA encoding light (L) chain of the antibody may be integrated intoseparate expression vectors, and then a host cell is co-transformed withthe resultant recombinant expression vectors. Alternatively, both DNAencoding H-chain and DNA encoding L-chain of the antibody may beintegrated together into a single expression vector, and then a hostcell is transformed with the resultant recombinant expression vector(for example, WO 94/11523).

The antibody gene can be expressed by known methods. For the expressionin a mammalian cell, a conventional useful promoter, the antibody geneto be expressed and a poly(A) signal (located downstream to the 3′ endof the antibody gene) may be operably linked. For example, as the usefulpromoter/enhancer system, a human cytomegalovirus immediate earlypromoter/enhancer system may be used.

Other promoter/enhancer systems, for example, those derived from viruses(e.g., retrovirus, polyoma virus, adenovirus and simian virus 40 (SV40))and those derived from mammalian cells (e.g., human elongation factor 1alpha (HEF1 alpha)), may also be used for the expression of the antibodyin the present invention.

When SV40 promoter/enhancer system is used, the gene expression may beperformed readily by the method of Mulligan et al. (Nature (1979) 277,108-14.). When HEF1 alpha promoter/enhancer system is used, the geneexpression may be performed readily by the method of Mizushima et al.(Nucleic Acids Res. (1990) 18, 5322.).

For the expression in E. coli, a conventional useful promoter, a signalsequence for secreting the antibody of interest and the antibody genemay be operably linked. As the promoter, lacZ promoter or araB promotermay be used. When lacZ promoter is used, the gene expression may beperformed by the method of Ward et al. (Nature (1098) 341, 544-6; FASBEJ. (1992) 6, 2422-7.), while when araB promoter is used, the geneexpression may be performed by the method of Better et al. (Science(1988) 240, 1041-3.).

With respect to the signal sequence for secretion of the antibody, whenthe antibody of interest is intended to be secreted in a periplasmicspace of the E. coli, pelB signal sequence (Lei, S. P. et al., J.Bacteriol. (1987) 169, 4379-83.) may be used. The antibody secreted intothe periplasmic space is isolated and then refolded so that the antibodytakes an appropriate configuration.

The replication origin derived from viruses (e.g., SV40, polyoma virus,adenovirus, bovine papilloma virus (BPV)) or the like may be used. Inorder to increase the gene copy number in the host cell system, theexpression vector may further contain a selective marker gene, such asan aminoglycoside phosphotranferase (APH) gene, a thymidine kinase (TK)gene, an E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt)gene and a dihydrofolate reductase (dhfr) gene.

For the production of the antibody used in the present invention, anyexpression system including eukaryotic and prokaryotic cell systems maybe used. The eukaryotic cell includes established cell lines of animals(e.g., mammals, insects, molds and fungi, yeast). The prokaryotic cellincludes bacterial cells such as E. coli cells. It is preferable thatthe antibody used in the present invention be expressed in a mammaliancell, such as a CHO, COS, myeloma, BHK, Vero and HeLa cell.

Next, the transformed host cell is cultured in vitro or in vivo toproduce the antibody of interest. The cultivation of the host cell maybe performed by any known method. The culture medium that can be usedherein may be DMEM, MEM, RPMI 1640 or IMDM medium. The culture mediummay contain a serum supplement, such as fetal calf serum (FCS).

In the production of the recombinant antibody, besides theabove-mentioned host cells, a transgenic animal may also be used as ahost. For example, the antibody gene is inserted into a predeterminedsite of a gene encoding a protein inherently produced in the milk of ananimal (e.g., beta-casein) to prepare a fusion gene. A DNA fragmentcontaining the antibody gene-introduced fusion gene is injected into anembryo of a non-human animal, and the embryo is then introduced into afemale animal. The female animal having the embryo therein bears atransgenic non-human animal. The antibody of interest is secreted in themilk from the transgenic non-human animal or a progeny thereof. For thepurpose of increasing the amount of the antibody-containing milk, anappropriate hormone may be administered to the transgenic animal (Ebert,K. M. et al., Bio/Technology (1994) 12, 699-702.).

The antibody expressed and produced as described above may be isolatedfrom the cells or the host animal body and purified. The isolation andpurification of the antibody used in the present invention may beperformed on an affinity column. Other methods conventionally used forthe isolation and purification of an antibody may be also be used; thusthe method is not particularly limited. For example, variouschromatographies, filtration, ultrafiltration, salting out and dialysismay be used singly or in combination to isolate and purify the antibodyof interest (Antibodies A Laboratory Manual. Ed. Harlow, David Lane,Cold Spring Harbor Laboratory, 1988).

(2) Chimeric Antibody and Humanized Antibody

In the present invention, an artificially modified recombinant antibodymay be used, including a chimeric antibody and a humanized antibody.These modified antibodies can be prepared by any known method. Forexample, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81: 6851-5;Neuberger et al., 1984, Nature, 312: 604-8; Takeda et al., 1985, Nature,314: 452-4.) can be used. A chimeric antibody is a molecule in whichdifferent portions are derived from different animal species, such asthose having a variable region derived from a murine mAb and a humanimmunoglobulin constant region, e.g., “humanized antibodies”.

A chimeric antibody according to the present invention can be preparedby ligating the DNA encoding the antibody V-region to DNA encoding ahuman antibody C-region, integrating the ligation product into anexpression vector, and introducing the resultant recombinant expressionvector into a host to produce the chimeric antibody.

A humanized antibody is also referred to as “reshaped human antibody”,in which the complementarity determining regions (CDRs) of an antibodyof a non-human mammal (e.g., a mouse) are grafted to those of a humanantibody. The general genetic recombination procedure for producing suchhumanized antibody is also known (for example, EP 125023; WO 96/02576.).

Specifically, a DNA sequence in which mouse antibody CDRs are ligatedthrough framework regions (FRs) is designed, and synthesized by a PCRmethod using several oligonucleotides as primers which were designed tohave regions overlapping to the terminal regions of the CDRs and theFRs. The resultant DNA is ligated to DNA encoding the human antibodyC-region, and the ligation product is integrated into an expressionvector. The resultant recombinant expression vector is introduced into ahost, thereby producing the humanized antibody (for example, WO96/02576).

The FRs ligated through the CDRs are selected so that the CDRs can forma functional antigen binding site. If necessary, an amino acid(s) in theFRs of the antibody V-region may be replaced so that the CDRs of thereshaped human antibody can form an appropriate antigen binding site(Sato, K. et al., Cancer Res. (1993) 53, 851-6.).

The chimeric antibody is composed of V-regions derived from a non-humanmammal antibody and C-regions derived from a human antibody. Thehumanized antibody is composed of CDRs derived from a non-human mammalantibody and FRs and C-regions derived from a human antibody. Thehumanized antibody may be useful for clinical use, because theantigenicity of the antibody against a human body is reduced.

A specific example of a chimeric antibody or a humanized antibody usedin the present invention is an antibody in which the CDRs are derivedfrom the mouse monoclonal antibody 92-13 or an antibody in which theCDRs are derived from the mouse monoclonal antibody 93-22. The methodfor producing such chimeric antibodies and humanized antibodies aredescribed below.

To clone DNA comprising a nucleotide sequence coding for V region of theanti-FZD10 mouse monoclonal antibody, mRNA can be isolated fromhybridomas and each cDNA in the V regions of L and H chains can besynthesized with the use of a reverse transcriptase as described above.In the synthesis of cDNA, Oligo-dT primer or other appropriate primerwhich hybridizes to L or H chain C region may be used. For example, butnot limited to, CH1 (IgG2a) primer having the nucleotide sequence asshown in SEQ ID NO: 3 for H chain V region and CL1 (kappa) primer havingthe nucleotide sequence as shown in SEQ ID NO: 4 for L chain V regioncan be used.

Amplification of cDNA of both L and H chains can be performed by PCR(polymerase chain reaction) using a commercially available kit (forexample, GeneRacer™ kit from Invitrogen) or using a known methodincluding 5′-RACE method (Frohman, M. A. et al., Proc. Natl. Acad. Sci.USA, 85, 8998-9002, 1988; Belyaysky, A. et al., Nucleic Acids Res., 17,2919-32, 1989.).

The specific primers for amplifying DNA for V regions of the mousemonoclonal antibody 92-13 include primers having the nucleotidesequences shown in SEQ ID NOs: 5 and 6 for H chain V region and primershaving the nucleotide sequences shown in SEQ ID NOs: 7 and 8 for L chainV region. Using these primers, a DNA encoding H chain V region having anamino acid sequence as shown in SEQ ID NO: 13 and a DNA encoding L chainV region having an amino acid sequence as shown in SEQ ID NO: 21 can beamplified. The specific primers for amplifying DNA for V regions of themouse monoclonal antibody 93-22 include primers having the nucleotidesequences shown in SEQ ID NOs: 53 and 54 for H chain V region andprimers having the nucleotide sequences shown in SEQ ID NOs: 55 and 56for L chain V region. Using these primers, a DNA encoding H chain Vregion having an amino acid sequence as shown in SEQ ID NO: 29 and a DNAencoding L chain V region having an amino acid sequence as shown in SEQID NO: 37 can be amplified.

Then, the amplified products are subjected to agarose gelelectrophoresis according to conventional procedures, and DNA fragmentsof interest are excised, recovered, purified and ligated to a vectorDNA.

The obtained DNA and vector DNA can be ligated using a known ligationkit to construct a recombinant vector. A vector DNA may be prepared in aknown method: J. Sambrook, et al., “Molecular Cloning”, Cold SpringHarbor Laboratory Press, 1989. The vector DNA is digested withrestriction enzyme(s), and the nucleotide sequence of a desired DNA canbe determined by a known method or using an automated sequencer.

Once DNA fragments coding for L and H chain V regions of mousemonoclonal antibody (hereinafter L or H chain of an antibody maysometimes be referred to as “mouse L or H chain” for mouse antibodiesand “human L or H chain” for human antibodies) are cloned, the DNAscoding for mouse V regions and DNAs coding for human antibody constantregions are ligated and expressed to yield chimeric antibodies.

A standard method for preparing chimeric antibodies involves ligating amouse leader sequence and V region sequence present in a cloned cDNA toa sequence coding for a human antibody C region already present in anexpression vector of a mammalian cell. Alternatively, a mouse leadersequence and V region sequence present in a cloned cDNA are ligated to asequence coding for a human antibody C region followed by ligation to amammalian cell expression vector.

The polypeptide comprising human antibody C region can be any of H or Lchain C regions of human antibodies, including, for example, C gamma 1,C gamma 2, C gamma 3 or C gamma 4 for human H chains or C lambda or Ckappa for L chains.

To prepare a chimeric antibody, two expression vectors are firstconstructed; that is, an expression vector containing DNAs coding formouse L chain V region and human L chain C region under the control ofan expression control element such as an enhancer/promoter system, andan expression vector containing DNAs coding for mouse H chain V regionand human H chain C region under the control of an expression controlelement such as an enhancer/promoter system, are constructed. Then, hostcells such as mammalian cells (for example, COS cell) are cotransformedwith these expression vectors and the transformed cells are cultivatedin vitro or in vivo to produce a chimeric antibody: see, for example,WO91/16928.

Alternatively, the mouse leader sequence present in the cloned cDNA andDNAs coding for mouse L chain V region and human L chain C region aswell as the mouse leader sequence and DNAs coding for mouse H chain Vregion and human H chain C region are introduced into a singleexpression vector (see, for example, WO94/11523) and said vector is usedto transform a host cell; then, the transformed host is cultured in vivoor in vitro to produce a desired chimeric antibody.

The vector for the expression of H chain of a chimeric antibody can beobtained by introducing cDNA comprising a nucleotide sequence coding formouse H chain V region (hereinafter referred to also as “cDNA for Hchain V region”) into a suitable expression vector containing thegenomic DNA comprising a nucleotide sequence coding for H chain C regionof human antibody (hereinafter referred to also as “genomic DNA for Hchain C region”) or cDNA coding for said region (hereinafter referred toalso as “cDNA for H chain C region”). The H chain C region includes, forexample, C gamma 1, C gamma 2, C gamma 3 or C gamma 4 regions.

The expression vectors having the genomic DNA coding for H chain Cregion, in particular, those coding for C gamma 1 region, include, forexample, HEF-PMh-g gamma 1 (WO92/19759) and DHER-INCREMENT E-RVh-PM1-f(WO92/19759). Alternatively, human constant region library can beprepared using cDNA from human PBMC (peripheral blood mononuclear cells)as described previously (Liu, A. Y. et al., Proc. Natl. Acad. Sci. USA,Vol. 84, 3439-43, 1987; Reff, M. E. et al., Blood, Vol. 83, No. 2,435-45, 1994).

When cDNA coding for mouse H chain V region is inserted into theseexpression vectors, an appropriate nucleotide sequence can be introducedinto said cDNA through PCR method. For instance, PCR may be effectedusing a PCR primer which is designed such that said cDNA has arecognition sequence for a suitable restriction enzyme at its 5′-end andKozak consensus sequence immediately before the initiation codon thereofso as to improve the transcription efficiency, as well as a PCR primerwhich is designed such that said cDNA has a recognition sequence for asuitable restriction enzyme at its 3′-end and a splice donor site forproperly splicing the primary transcription products of the genomic DNAto give a mRNA, to introduce these appropriate nucleotide sequences intothe expression vector.

The thus constructed cDNA coding for mouse H chain V region is treatedwith a suitable restriction enzyme(s), then it is inserted into saidexpression vector to construct a chimeric H chain expression vectorcontaining the genome DNA coding for H chain C region (C gamma 1region).

The thus constructed cDNA coding for mouse H chain V region is treatedwith a suitable restriction enzyme(s), ligated to cDNA coding for said Hchain C region C gamma 1, and inserted into an expression vector such aspQCXIH (Clontech) to construct an expression vector containing the cDNAcoding for a chimeric H chain.

The vector for the expression of L chain of a chimeric antibody can beobtained by ligating a cDNA coding for mouse L chain V region and agenomic DNA or cDNA coding for L chain C region of a human antibody andintroducing into a suitable expression vector. The L chain C regionincludes, for example, kappa chain and lambda chain.

When an expression vector containing cDNA coding for mouse L chain Vregion is constructed, appropriate nucleotide sequences such as arecognition sequence or Kozak consensus sequence can be introduced-intosaid expression vector through PCR method.

The entire nucleotide sequence of cDNA coding for human L lambda chain Cregion may be synthesized by a DNA synthesizer and constructed throughPCR method. The human L lambda chain C region is known to have at least4 different isotypes and each isotype can be used to construct anexpression vector.

The constructed cDNA coding for human L lambda chain C region and theabove constructed cDNA coding for mouse L chain V region can be ligatedbetween suitable restriction enzyme sites and inserted into anexpression vector such as pQCXIH (Clontech), to construct an expressionvector containing cDNA coding for a L lambda chain of a chimericantibody.

The DNA coding for human L kappa chain C region to be ligated to the DNAcoding for mouse L chain V region can be constructed from, for example,HEF-PM1k-gk containing the genomic DNA (see WO92/19759). Alternatively,human constant region library can be prepared using cDNA from human PBMC(peripheral blood mononuclear cells) as described previously (Liu, A. Y.et al., Proc. Natl. Acad. Sci. USA, Vol. 84, 3439-43, 1987; Reff, M. E.et al., Blood, Vol. 83, No. 2, 435-45, 1994).

Recognition sequences for suitable restriction enzymes can beintroduced, through PCR method, into 5′- and 3′-ends of DNA coding for Lkappa chain C region, and the DNA coding for mouse L chain V region asconstructed above and the DNA coding for L kappa chain C region can beligated to each other and inserted into an expression vector such aspQCXIH (Clontech) to construct an expression vector containing cDNAcoding for L kappa chain of a chimeric antibody.

In order to make a humanized antibody in which CDR of a mouse monoclonalantibody is grafted to a human antibody, it is desirable that thereexists a high homology between FR of the mouse monoclonal antibody andFR of the human antibody. Accordingly, a comparison is made between Vregions of H and L chains of mouse anti-FZD10 monoclonal antibody andthe V regions of all the known antibodies whose structures have beenelucidated with the use of Protein Data Bank. Further, they aresimultaneously compared with the human antibody subgroups (HSG: Humansubgroup) classified by Kabat et al. based on the length of antibody FR,the homology of amino acids, and the like: Kabat, E. A. et al, US Dep,Health and Human Services, US Government Printing Offices, 1991.

The first step for designing DNA coding for a humanized antibody Vregion is to select a human antibody V region as a basis for thedesigning. For example, FR of a human antibody V region having ahomology of higher than 80% with FR of a mouse antibody V region can beused in the production of a humanized antibody.

In the humanized antibody, the C region and the framework (FR) regionsof the V region of said antibody are originated from human and thecomplementarity determining regions (CDR) of the V region are originatedfrom mouse. A polypeptide comprising the V region of the humanizedantibody can be produced in the manner called CDR-grafting by PCR methodso long as a DNA fragment of a human antibody would be available as atemplate. The “CDR-grafting” refers to a method wherein a DNA fragmentcoding for a mouse-derived CDR is made and replaced for the CDR of ahuman antibody as a template.

If a DNA fragment of a human antibody to be used as a template is notavailable, a nucleotide sequence registered in a database may besynthesized in a DNA synthesizer and a DNA for a V region of a humanizedantibody can be produced by the PCR method. Further, when only an aminoacid sequence is registered in the database, the entire nucleotidesequence may be deduced from the amino acid sequence on the basis ofknowledge on the codon usage in antibodies as reported by Kabat, E. A.et al. in US Dep. Health and Human Services, US Government PrintingOffices, 1991. This nucleotide sequence is synthesized in a DNAsynthesizer and a DNA of a humanized antibody V region can be preparedby PCR method and introduced into a suitable host followed by expressionthereof to produce the desired polypeptide.

General procedures of CDR-grafting by PCR method are described belowwhen a DNA fragment of a human antibody as a template is available.

First, mouse derived DNA fragments corresponding to respective CDRs aresynthesized. CDRs 1 to 3 are synthesized on the basis of the nucleotidesequences of the previously cloned mouse H and L chain V regions. Forexample, when a humanized antibody is produced based on the mousemonoclonal antibody 92-13, CDR sequences of chain V region can be theamino acid sequences as shown in SEQ ID NOs: 15 (VH CDR1), 17 (VH CDR2)and 19 (VH CDR3); and CDR sequences of L chain V region can be the aminoacid sequences as shown in SEQ ID NOs: 23 (VL CDR1), 25 (VL CDR2) and 27(VL CDR3). When a humanized antibody is produced based on the mousemonoclonal antibody 93-22, CDR sequences of H chain V region can be theamino acid sequences as shown in SEQ ID NOs: 31 (VH CDR1), 33 (VH CDR2)and 35 (VH CDR3); and CDR sequences of L chain V region can be the aminoacid sequences as shown in SEQ ID NOs: 39 (VL CDR1), 41 (VL CDR2) and 43(VL CDR3).

The DNA for H chain V region of a humanized antibody may be ligated toDNA for any human antibody H chain C region, for example, human H chainC gamma 1 region. As mentioned above, the DNA for H chain V region maybe treated with a suitable restriction enzyme and ligated to a DNAcoding for a human H chain C region under an expression control elementsuch as an enhancer/promoter system to make an expression vectorcontaining DNAs for a humanized H chain V region and a human H chain Cregion.

The DNA for L chain V region of a humanized antibody may be ligated toDNA for any human antibody L chain C region, for example, human L chainC lambda region. The DNA for L chain V region may be treated with asuitable restriction enzyme and ligated to a DNA coding for a human Llambda chain C region under an expression control element such as anenhancer/promoter system to make an expression vector containing DNAscoding for a humanized L chain V region and a human L lambda chain Cregion.

The DNA coding for H chain V region of a humanized antibody and a humanH chain C region and the DNA coding for a humanized L chain V region andhuman L chain C region may also be introduced into a single expressionvector such as that disclosed in WO94/11523, said vector may be used totransform a host cell, and the transformed host may be cultivated invivo or in vitro to produce a desired humanized antibody.

To produce a chimeric or humanized antibody, two expression vectors asabove mentioned should be prepared. Thus, with respect to a chimericantibody, an expression vector comprising a DNA coding for a mouse Hchain V region and a human H chain C region under the control of anexpression control element such as an enhancer/promoter, and anexpression vector comprising a DNA coding for a mouse L chain V regionand a human L chain C region under the control of an expression controlelement are constructed. With respect to a humanized antibody, anexpression vector comprising a DNA coding for a humanized H chain Vregion and a human H chain C region under the control of an expressioncontrol element, and an expression vector comprising a DNA coding for ahumanized L chain V region and a human L chain C region under thecontrol of an expression control element are constructed.

Then, a host cell such as a mammalian cell (for example, COS cell) maybe cotransformed with these expression vectors and the resultingtransformed cell may be cultured in vitro or in vivo to produce thechimeric or humanized antibody (see, for example, WO91/16928).

Alternatively, a DNA coding for H chain V and C regions and a DNA codingfor L chain V and C regions may be ligated to a single vector andtransformed into a suitable host cell to produce an antibody. Thus, inthe expression of a chimeric antibody, a DNA coding for a mouse leadersequence present in the cloned cDNA, a mouse H chain V region and ahuman H chain C region as well as a DNA coding for a mouse leadersequence, a mouse L chain V region and a human L chain C region, can beintroduced into a single expression vector such as one disclosed in e.g.WO94/11523. In the expression of a humanized antibody, a DNA coding fora humanized H chain V region and a human H chain C region and a DNAcoding for a humanized L chain V region and a human L chain C region maybe introduced into a single expression vector such as one disclosed ine.g. WO94/11523. Such a vector is used to transform a host cell and thetransformed host is cultured in vivo or in vitro to produce a chimericor humanized antibody of interest.

Any expression system may be used to produce the chimeric or humanizedantibody against FZD10 protein according to the present invention. Forexample, eukaryotic cells include animal cells such as establishedmammalian cell lines, fungal cells, and yeast cells; prokaryotic cellsinclude bacterial cells such as Escherichia coli. Preferably, thechimeric or humanized antibody of the present invention is expressed ina mammalian cell such as COS or CHO cell.

Any conventional promoters useful for the expression in mammalian cellsmay be used. For example, human cytomegalovirus (HCMV) immediate earlypromoter is preferably used. In addition, promoters for gene expressionin mammalian cells may include virus promoters, such as those ofretrovirus, polyoma virus, adenovirus and simian virus (SV) 40, andmammalian cell derived promoters, such as those of human polypeptidechain elongation factor-1 alpha (HEF-1 alpha). For example, SV40promoter may be readily used according to Mulligan et al. method(Nature, 277, 108-14, 1979); Mizushima, S. et al. method (Nucleic AcidsResearch, 18, 5322, 1990) may be easily used with HEF-1 alpha promoter.

Replication origin includes those derived from SV40, polyoma virus,adenovirus or bovine papilloma virus (BPV). Further, the expressionvector may comprise a gene for phosphotransferase APH(3′) II or I (neo),thymidine kinase (TK), E. coli xanthine-guaninephosphoribosyltransferase (Ecogpt) or dihydrofolate reductase (DHFR) asa selective marker for increasing the gene copy number in a host cellsystem.

The chimeric or humanized antibody of interest which is thus produced byculturing the transformant transformed with a DNA coding for thechimeric or humanized antibody may be isolated from the cell and thenpurified.

The isolation and purification of the chimeric or humanized antibody ofinterest may be carried out by using a protein A agarose column, but mayalso be performed by any methods used in isolation and purification of aprotein and thus is not limited. For instance, a chromatography,ultrafiltration, salting out and dialysis may optionally be selected orcombined to isolate and purify the chimeric or humanized antibody.

After isolating the chimeric antibody or humanized antibody, theconcentration of the resulting purified antibody can be determined byELISA.

The determination of the antigen-binding activity or other activitiesincluding binding activity to a normal cell of the chimeric antibody orhumanized antibody may be performed by any known methods (Antibodies ALaboratory Manual, Ed. Harlow, David Lane, Cold Spring HarborLaboratory, 1988).

As the method for the determination of the antigen-binding activity ofan antibody, techniques such as ELISA (enzyme-linked immunosorbentassay), EIA (enzyme immunoassay), RIA (radioimmunoassay) or fluorescentassay may be employed.

(3) Antibody Fragment and Modified Antibody

The antibody used in the present invention may be any fragment thereofor a modified antibody, as long as it can bind to FZD10 protein andinhibit its activity. For example, the fragment of the antibody includesFab, F(ab′)₂, Fv, or a single chain Fv (scFv) composed of a H-chain Fvfragment or a L-chain Fv fragment linked together through a suitablelinker. Specifically, such antibody fragments can be produced bycleaving the antibody with an enzyme (e.g., papain, pepsin) intoantibody fragments, or by constructing a gene encoding the antibodyfragment and inserting the gene into an expression vector andintroducing the resultant recombinant expression vector into a suitablehost cell, thereby expressing the antibody fragment (see, for example,Co, M. S., et al., J. Immunol. (1994), 152, 2968-76; Better, M. &Horwitz, A. H., Methods in Enzymology (1989), 178, 476-96, AcademicPress, Inc; Pluckthun, A. & Skerra, A., Methods in Enzymology (1989)178, 497-515, Academic Press, Inc; Lamoyi, E., Methods in Enzymology(1989) 121, 652-63; Rousseaux, J. et al., Methods in Enzymology (1989)121, 663-9; and Bird, R. E. et al., Trends Biotechnol. (1991) 9, 132-7).Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246: 1275-81) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

A scFv can be produced by ligating the H-chain V-region to the L-chainV-region through a linker, preferably a peptide linker (Huston, J. S. etal., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-83). The H-chainV-region and the L-chain V-region in the scFv may be derived from anyone of the antibodies described herein. The peptide linker which bindsthe V-regions may be any single chain peptide, for example, of 12-19amino acid residues.

As a modified antibody, for example, anti-FZD10 antibody or fragmentthereof conjugated to any molecule (e.g., polyethylene glycol) may alsobe used. Such modified antibodies are also encompassed in the “antibody”of the present invention. The modified antibodies can be prepared bychemical modifications of the antibodies. The chemical modificationtechniques suitable for this purpose have already been established inthe art.

2. Therapeutic Uses

Described below are methods and pharmaceutical compositions for treatingand/or preventing FZD10-associated disease using the antibody of thepresent invention. The outcome of a treatment is to at least produce ina treated subject a healthful benefit, which in the case of tumors,includes but is not limited to remission of the tumors, palliation ofthe symptoms of the tumors, and control of metastatic spread of thetumors.

Specifically, the method for treating and/or preventing FZD10-associateddisease in a subject according to the present invention comprisesadministering to a subject in need thereof the antibody or the fragmentdescribed above.

The term “subject” herein refers to a subject who has suffered fromFZD10-associated disease and also a subject suspected to haveFZD10-associated disease. The subject in the present invention may beanimals including mammals and avian animals. For example, mammals mayinclude humans, mice, rats, monkeys, rabbits, and dogs.

The term “FZD10-associated disease” herein refers to a diseaseassociated with the over-expression of FZD10 protein. Specifically,FZD10-associated diseases include, but are not limited to, synovialsarcoma (SS), colorectal cancer, gastric cancer, chronic myeloidleukemia (CML), and acute myeloid leukemia (AML).

The antibody or fragment thereof described herein can specifically bindto FZD10 protein, so when the antibody or fragment thereof isadministered to a subject, it binds to FZD10 protein in the subject andthe activity of FZD10 protein may be inhibited. Alternatively, when theantibody or fragment thereof may be conjugated with a therapeutic moietyand administered to a subject, it is delivered to a region thatexpresses FZD10 protein (i.e. suffered region) in a subject and thetherapeutic moiety can be selectively delivered to the suffered regionand acted thereon. Such therapeutic moiety may be any therapeutics thatare known or will be developed for having a therapeutic efficacy onFZD10-associated disease and includes, but not limited to, aradioisotope label and chemotherapeutic agent. A radioisotope labelwhich can be used as therapeutics can be selected depending on a varietyof elements including β-ray energy and its emission efficiency, thepresence or absence of γ-ray emitted, its energy and emissionefficiency, physical half-life, and labeling procedure. Generally, theradioisotope label based on yttrium (such as ⁹⁰Y) and iodine (such as¹²⁵I and ¹³¹I) may be used. A chemotherapeutic agent may be any agentthat is known or will be developed for treating FZD10-associated diseaseand includes, but not limited to, methotrexate, taxol, mercaptopurine,thioguanine, cisplatin, carboplatin, mitomycin, bleomycin, doxorubicin,idarubicin, daunorubicin, dactinomycin, vinblastine, vincristine,vinorelbine, paclitaxel, and docetaxel. The antibody or fragment thereofdescribed herein can selectively bind to FZD10 protein and not bind to anormal cell, so side effect which is caused by the antibody or fragmentthereof, or radioisotope or chemotherapeutic agent can be effectivelyavoided and therefore the therapeutic potency may be high.

The antibody or fragment thereof described herein can be administered toa subject at effective doses to treat or prevent the FZD10-associateddisease. An effective dose refers to that amount of an antibody or afragment thereof sufficient to result in a healthful benefit in thetreated subject. Formulations and methods of administration that can beemployed when the pharmaceutical composition contains an antibody of thepresent invention are described below.

Pharmaceutical compositions for use in accordance with the presentinvention can be formulated in conventional manner using one or morepharmaceutically acceptable carriers or excipients.

The antibodies or fragments thereof can be formulated for parenteraladministration e., intravenous or intramuscular) by injection, via, forexample, bolus injection or continuous infusion. Formulations forinjection can be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions cantake such forms as suspensions, solutions, or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the antibody can bein lyophilized powder form for constitution with a suitable vehicle,e.g., sterile pyrogen-free water, before use.

Toxicity and therapeutic efficacy of the antibody or fragment, or thetherapeutic moiety conjugated thereto can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD/ED.

Antibodies or therapeutic moieties that exhibit large therapeuticindices are preferred. While antibodies or moieties that exhibit toxicside effects can be used, care should be taken to design a deliverysystem that targets such antibodies or moieties to the site of affectedtissue in order to minimize potential damage to uninfected cells and,thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosages for use in humans. The dosage ofsuch antibodies lies preferably within a range of circulating plasmaconcentrations that include the ED50 with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed, the route of administration utilized and types and amounts ofthe therapeutic moiety conjugated. For any antibody used in the methodof the invention, the effective dose can be estimated initially fromcell culture assays. A dose can be formulated in animal models toachieve a circulating plasma concentration range that includes the IC50(i.e., the concentration of the test antibody that achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

While depending on the conditions and age of the subject and/oradministration route, one skilled in the art can select an appropriatedose of the pharmaceutical composition of the present invention. Forexample, the pharmaceutical composition of the present invention isadministered in an amount such that the antibody according to thepresent invention is administered to the subject in a day in an amountof about 3 to about 15 μg per kg body weight of subject, and preferablyof about 10 to about 15 μg per kg body weight of subject. Theadministration interval and times can be selected in consideration ofthe condition and age of the subject, administration route, and responseto the pharmaceutical composition. For example, the pharmaceuticalcomposition can be administered to the subject one to 5 times,preferably 1 times a day for 5 to 10 days.

The pharmaceutical composition can be administered systemically orlocally. It is preferably administered in a targeting delivery manner soas to deliver the active component to an affected site.

In particular embodiments, the methods and compositions of the presentinvention are used for the treatment or prevention of FZD10-associateddisease together with one or a combination of chemotherapeutic agentsincluding, but not limited to, methotrexate, taxol, mercaptopurine,thioguanine, cisplatin, carboplatin, mitomycin, bleomycin, doxorubicin,idarubicin, daunorubicin, dactinomycin, vinblastine, vincristine,vinorelbine, paclitaxel, and docetaxel.

With respect to radiation therapy, any radiation therapy protocol can beused depending upon the type of FZD10-associated disease to be treated.For example, but not by way of limitation, X-ray radiation can beadministered. Gamma ray emitting radioisotopes, such as radioactiveisotopes of radium, cobalt, and other elements may also be administeredto expose tissues.

In another embodiment, chemotherapy or radiation therapy isadministered, preferably at least an hour, five hours, 12 hours, a day,a week, a month, and more preferably several months (e.g., up to threemonths) subsequent to using the methods and compositions containing theantibody of the present invention. The chemotherapy or radiation therapyadministered prior to, concurrently with, or subsequent to the treatmentusing the methods and compositions according to the present inventioncan be administered by any method known in the art.

3. Diagnostic and Prognotic Uses

Antibodies directed against FZD10 protein or fragments thereof may alsobe used as diagnostics and prognostics, as described herein. Suchdiagnostics methods may used to detect the presence or absence ofFZD10-associated disease and the risk of having the disease. The methodfor diagnosis and/or prognosis of an FZD10-associated disease of thepresent invention comprises immunologically detecting or determining theFZD10 protein derived from the disease in a sample using an antibody ora fragment thereof according to the present invention. Specifically, amethod for diagnosis or prognosis of FZD10-associated disease or of apredisposition to develop the disease in a subject according to thepresent invention comprises:

(a) contacting a sample from the subject with an antibody against FZD10protein or a fragment thereof;

(b) detecting the FZD10 protein in the sample; and

(c) judging whether or not the subject suffers from or is at risk ofdeveloping the disease based on the relative abundance of the FZD10protein compared to a control.

The method for diagnosis and/or prognosis of the present invention canbe performed based on any procedures, as long as it is an assay using anantibody, i.e., an immunological assay. Thereby one can detect the FZD10protein using the antibody or a fragment thereof of the presentinvention as the antibody used in the assay. For example, the FZD10protein can be detected by using an immunohistochemical staining,immunoassay such as enzyme immunoassays (ELISA and EIA),immunofluorescent assay, radioimmunoassay (RIA), or Western blotting.

A sample to be tested in the method for diagnosis and/or prognosis ofFZD10-associated disease of the present invention is not specificallylimited, as long as it is a biological sample that may contain the FZD10protein derived from the FZD10-associated disease. Examples of thesample include extract of a cell or organ, and tissue sections, as wellas blood, sera, plasma, lymphocyte cultivated supernatant, urine, spinalfluid, saliva, sweat, and ascites. The abundance of the FZD10 protein asdetermined in samples such as tumor tissue, tumor biopsy, and metastasistissue by using the antibody or a fragment thereof of the presentinvention is specifically useful as an index of an FZD10-associateddisease.

For example, antibodies and fragments thereof described herein may beused to quantitatively or qualitatively detect the FZD10 protein. Theantibodies (or fragment thereof) of the present invention may,additionally, be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of FZD10 protein. Insitu detection may be accomplished by removing a histological samplefrom a subject, such as paraffin-embedded sections of tissues (such assurgical specimens) and applying thereto a labeled antibody of thepresent invention. The antibody (or fragment thereof) is preferablyapplied by overlaying a sample with the labeled antibody (or fragmentthereof). Using the present invention, those skilled in the art willreadily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

Immunoassays for FZD10 protein will typically comprise incubating asample from a subject to be examined, such as a biological fluid, atissue extract, freshly harvested cells, or lysates of cells that havebeen incubated in cell culture, in the presence of a detectably labeledantibody of the present invention, and detecting the bound antibody byany of a number of techniques well-known in the art.

The sample may be brought into contact with and immobilized onto a solidphase support or carrier such as nitrocellulose, or another solidsupport which is capable of immobilizing cells, cell particles, orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled antibody againstFZD10. The solid phase support may then be washed with the buffer asecond time to remove unbound antibody. The amount of bound label on thesolid support may then be detected by conventional means.

The term “solid phase support or carrier” means any support capable ofbinding an antigen or an antibody. Those skilled in the art will knowmany suitable carriers for binding antibodies or antigens, or will beable to ascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-FZD10 antibody may bedetermined according to well-known methods. Those skilled in the artwill be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation.

To detect a reaction between the antibody (or its fragment) of thepresent invention and the FZD10 protein derived from an FZD10-associateddisease affected site in a sample easily, the reaction can be directlydetected by labeling the antibody of the present invention or indirectlydetected by using a labeled secondary antibody. The latter indirectdetection procedure, such as a sandwich assay or competitive assay ofELISA, is preferably used in the method of the present invention forbetter sensitivity.

Examples of labels for use herein are as follows. Peroxidases (PODs),alkaline phosphatases, β-galactosidase, urease, catalase, glucoseoxidase, lactate dehydrogenase, amylases, and biotin-avidin complexescan be used in an enzyme immunoassay. Fluorescein isothiocyanate (FITC),tetramethylrhodamine isothiocyanate (TRITC), substituted rhodamineisothiocyanate, dichlorotriazine isothiocyanate and Alexa488 can be usedin an immunofluorescent assay. Tritium, iodine (such as ¹²⁵I, and ¹³¹I),and indium (such as ¹¹¹In) can be used in a radioimmunoassay.NADH-FMNH₂-luciferase assay, luminol-hydrogen peroxide-POD system,acridinium esters, and dioxetane compounds can be used in animmunoluminescent assay.

The label can be attached to the antibody according to a conventionalprocedure. For example, the label can be attached to the antibody by aglutaraldehyde method, maleimide method, pyridyl disulfide method, orperiodate method in the enzyme immunoassay, and by a chloramine T methodor Bolton-Hunter method in the radioimmunoassay.

The assay can be performed according to a known procedure (Ausubel, F.M. et al. Eds., Short Protocols in Molecular Biology, Chapter 11“Immunology” John Wiley & Sons, Inc. 1995).

For example, when the antibody of the present invention is directlylabeled with the label described above, the sample is brought intocontact with the labeled antibody to thereby form a complex between theFZD10 protein and the antibody. Then, unbound labeled antibody isseparated, and the level of the FZD10 protein in the sample can bedetermined based on the amount of the bound labeled antibody or that ofthe unbound labeled antibody.

When a labeled secondary antibody is used, the antibody of the presentinvention is allowed to react with the sample in a primary reaction, andthe resulting complex is allowed to react with the labeled secondaryantibody in a secondary reaction. The primary reaction and the secondaryreaction can be performed in reverse order, concurrently with someinterval of time therebetween. The primary and secondary reactions yielda complex of [FZD10 protein]-[the antibody of the invention]-[thelabeled secondary antibody] or a complex of [the antibody of theinvention]-[FZD10 protein]-[the labeled secondary antibody]. Unboundlabeled secondary antibody is then separated, and the level of the FZD10protein in the sample can be determined based on the abundance of thebound labeled secondary antibody or that of the unbound labeledsecondary antibody.

According to another embodiment, the antibody of the present inventionis labeled with a radioisotope or a fluorescent label, and the labeledantibody is parenterally administered to a subject. Thus, thelocalization of a primary tumor and the related metastasized tumor ofFZD10-associated disease can be rapidly found in a non-invasive manner.Such a diagnosis method is known as tumor in vivo imaging, and oneskilled in the art can easily understand the procedures thereof. Thelabeled antibody can be administered to the subject systemically orlocally, preferably through a parenteral route such as intravenousinjection, intramuscular injection, intraperitoneal injection, orsubcutaneous injection.

The antibodies according to the present invention specifically reactwith a FZD10 protein as mentioned above and can thereby be used in kitsfor diagnosis and/or prognosis of an FZD10-associated disease.

The kit for diagnosis and/or prognosis of the present inventioncomprises an antibody or a fragment thereof described herein. Bydetecting the FZD10 protein in a sample from a subject who is suspectedto suffer from an FZD10-associated disease with the use of the kit fordiagnosis and/or prognosis of the present invention, whether or not thesubject suffers from the FZD10-associated disease can be rapidly andeasily ascertained. Kits for diagnosis and/or prognosis of diseasesusing such immunological reactions have been widely known, and oneskilled in the art can easily select appropriate components other thanthe antibody. The kits for diagnosis and/or prognosis of the presentinvention can be used in any means, as long as it is a means forimmunoassay.

EXAMPLES

The present invention will be further illustrated by the followingnon-limiting examples.

Cell lines and tissue specimens used in the following examples wereprepared as described below. Specifically, cell lines derived fromsynovial sarcomas (HS-SY-2, YaFuSS, 1973/99, Fuji and SYO-1), coloncancers (LoVo, SNU-C4 and SNU-C5), HEK293 and COS7 cells were grown inmonolayers in appropriate media supplemented with 10% fetal bovine serumand 1% antibiotic/antimycotic solution, and maintained at 37° C. in aircontaining 5% CO₂. Primary synovial sarcoma (SS) samples were obtainedafter informed consent, and snap-frozen in liquid nitrogen immediatelyafter resection and stored at −80° C.

Example 1 Generation of anti-FZD10 Monoclonal Antibodies

(1) Generating Monoclonal Antibodies with Cell Immunization

Mouse anti-FZD10 monoclonal antibodies (Mabs) were generated byimmunizing four weeks old female Balb/c mice in their foot pads with2×10⁷ COS-7 cells transfected with 2×10⁷ of pCAGGS/neo-FZD10-myc/His(Medical and Biological Laboratories, Nagoya, Japan). Construction ofpCAGGS/neo-FZD10-myc/H is was reported previously (Nagayama, S., et al.(2005). Oncogene, 24, 6201-12.) and this expresses the entire codingsequence of FZD10 cDNA and Myc and His epitope tags at its C terminus.The mice had been immunized with Freund complete adjuvant (MitsubishiKagaku latron, Inc., Tokyo, Japan) in one day prior to the cellimmunization. Spleen cells from the immunized mice were harvested andfused with the myeloma cell line. The hybridomas were subcloned andassayed by Cell ELISA for the ability to secrete immunoglobulin thatbinds to the extracellular domain of FZD10 (amino acid residues 1-225 ofFZD10). For cell ELISA, COS-7 cells expressing FZD10-myc/His (the entirecoding sequence of FZD10 cDNA and Myc and His epitope tags at its Cterminus) were seeded into 96-well plates. Subsequently, 50 μl of theculture supernatants obtained from hybridomas were added to the plateand incubated for 30 minutes at room temperature. After washing thecells, goat anti-mouse IgG-POD (Medical and Biological Laboratories,Nagoya, Japan) was added at 1:10000 dilution, incubated for 30 minutesat room temperature. Bound antibodies were detected at OD₄₅₀₋₆₂₀ nm.Positive clones were further analyzed for specific binding activity.These clones includes: clones 39-2 and 39-10 (disclosed inWO2005/004912, referred to as 5F2) as well as 92-13 and 93-22. All Mabswere of the IgG2a isotype as determined by means of the IsoStrip MouseMonoclonal antibody isotyping kit (Roche). The Mabs were affinitypurified on protein G-sepharose for further characterization.

The hybridoma clone 93-22 producing mouse monoclonal antibody 93-22 wasdeposited by Shuichi Nakatsuru internationally at the IPOD InternationalPatent Organism Depository of the National Institute of AdvancedIndustrial Science and Technology (AIST Tsukuba Central 6, 1-1, Higashi1-chome, Tsukuba-shi, Ibaraki-Ken, 305-8566 Japan) as of Jun. 14, 2006under the deposit number of FERM BP-10620. Also, hybridoma clone 92-13producing mouse monoclonal antibody 92-13 was deposited by ShuichiNakatsuru internationally at the IPOD International Patent OrganismDepository of the National Institute of AIST as of Jun. 28, 2006 underthe deposit number of FERM BP-10628.

(2) Labeling Antibodies with Radionuclides

¹²⁵I-labeled Mabs were prepared by chloramine T method (Arano, Y., etal. (1999). Cancer Res, 59, 128-34.). 740 kBq/2 μl of Na¹²⁵I was addedto 10 μg of Mab in 100 μl of 0.3M sodium phosphate buffer. One μg ofchloramine-T in 3 μl of 0.3M sodium phosphate buffer was further added,incubated for 5 min at room temperature. Labeled antibody was purifiedusing Biospin column 6 (Bio-Rad).

For labeling Mabs with ¹¹¹In, 1 mg of Mab in 100 μl of 50 mM boratebuffer (pH8.5) was conjugated to isothiocyanato benzyldiethylenetriaminepentaacetic acid (SCN-BZ-DTPA; Macrocyclics) indimethylformamide at molar ratio 1:3. After incubation at 37° C. for 20hours, Mab conjugates were purified using Biospin column 6. 40 μl of¹¹¹In was incubated in 60 μl of 0.25M acetic acid buffer (pH5.5) andincorporated into 10 μg/μl of Mab-DTPA conjugates for one hour at roomtemperature. Labeled antibody was purified using Biospin column 6.

For generating ⁹⁰Y-conjugated 92-13, 92-13 was conjugated with DTPA tolysine residues. DTPA-92-13 was labeled with yttrium to a specificactivity 100 μCi/mg, and the immunoreactivity of the ⁹⁰Y-DTPA-92-13 wasapproximately 70%.

(3) Synthesis of Alexa647-Labeled Mabs.

Labeling Mabs with Alexa-Fluoro647 was carried out according tomanufacturer's instruction using Alexa647 Monoclonal Antibody LabelingKit (Molecular Probes, Eugene, Oreg.). The Alexa647 reactive dye has asuccinimidyl ester moiety that reacts with primary amines of proteins,and resulting Mabs-dye conjugates were purified by size exclusioncolumn.

Example 2 Binding Activities of Anti-FZD10 Monoclonal Antibodies

The present inventors applied two methods for evaluation of the bindingaffinity of mouse-monoclonal antibodies; flow cytometrical analysis withfluorescent dyes and radioactive measurement using ¹²⁵I.

(1) Flow Cytometry (FACS) Analysis

To investigate the cell-binding affinities of the four antibodies, 39-2and 39-10 (disclosed in WO2005/004912), 92-13 and 93-22, we performedflow cytometry (FACS) experiments. For flow cytometrical analysis withindirect fluorescence, suspensions of 5×10⁶ cells were incubated with 10μg/ml of Mabs or non-immunized mouse IgG (Beckman Coulter) for 30 min at4° C. After washing with PBS, 2 μg of fluorescent goat anti-mouse IgG(Alexa Fluor 488, Molecular Probes, Eugene, Oreg.) was added, and thecell suspension was incubated for 30 min at 4° C. for analysis byFACScan (Becton Dickinson, Franklin Lakes, N.J.). For directimmunofluorescence assays, cells were incubated with 2 μg ofAlexa488-Mabs in the presence or absence of excess amount (100 μg) ofnon-labeled Mabs for 30 min at 4° C. and subjected to analysis byFACScan.

In order to confirm the expression of FZD10 in cell lines, we performedRT-PCR. For RT-PCR experiments, total RNAs were extracted from celllines using TRIzol reagent (Invitrogen, Carlsbad, Calif., USA), and 3 μgaliquot of each total RNA was reversely transcribed. PCR amplificationwas performed using the cDNAs as templates with the following primers:5′-TATCGGGCTCTTCTCTGTGC-3′ (SEQ ID NO: 9) and 5′-GACTGGGCAGGGATCTCATA-3′(SEQ ID NO: 10) for FZD10 and 5′-TTAGCTGTGCTCGCGCTACT-3′ (SEQ ID NO: 11)and 5′-TCACATGGTTCACACGGCAG-3′ (SEQ ID NO: 12) for β2-microglobulin(β2MG), the internal control.

As shown in FIG. 1 a, all of four Mabs, 39-2, 39-10, 92-13 and 93-22bound to four FZD10-expressing SS cell lines, SYO-1, YafuSS, HS-SY-2,and Fuji in FZD10-dose dependent manner, but not bound to two celllines, 1973/99 and LoVo, in which no transcript of FZD10 was detected.Table 1 below indicates correlation between relative Mean fluorescentIntensities (MFI) of these Mabs and the expression levels of FZD10 shownin FIG. 1 b. In addition, particularly, we demonstrated that 92-13 and93-22 Mabs also bound to the SNU-C5 transfected with FZD10-myc/Hisconstruct, while no binding was detected with SNU-C5 cells transfectedwith empty vector (FIG. 1 c), suggesting specific binding of those 92-13and 93-22 Mabs against FZD10 protein.

TABLE 1 Binding of anti-FZD10 mAbs to human SS cell lines SYO-1, YaFuSS,HS-SY-II, Fuji, 1973/99 and human colon cancer cell line, LoVo. SYO-1YaFuss HS-SY-II Fuji 1973/99 LoVo 39-2 17.6 11.6 9.4 7.1 5.5 1.1 39-1018.4 11.8 9.9 6.9 4.9 1.0 92-13 4.7 3.0 3.0 1.3 0.9 1.0 93-22 3.3 2.72.4 1.1 1.0 1.1 The MFI of FZD10 is measured by flow cytometry asdescribed above.(2) Binding Activity Against Normal Blood Cells

To confirm whether those antibodies can be applied for clinical use, thepresent inventors further examined the binding activity of antibodiesagainst normal blood cells. To evaluate the non-specific bindingactivity of Mabs against normal blood cell, ¹²⁵I-labeled Mabs wereincubated with 100 μl of healthy fresh blood. After incubation for onehour at room temperature, the radioactivities of cell pellet weremeasured as described above.

The binding activity of ¹²⁵I-labeled 92-13 and 93-22 Mabs against normalhuman blood cells were undetectable in all of three individual donors,whereas those of 39-2 and 39-10 Mabs were detected in all of threeindividual donors (FIG. 1 d). These results were consistent with thoseof FACS analysis using human peripheral blood mononuclear cell (data notshown), suggesting clinical applicability of only 92-13 and 93-22antibodies with little possibility of adverse effect to SS patientsbecause of very specific binding affinity to the FZD10 molecule.Therefore, we focused on only 92-13 and 93-22 antibodies for furtheranalysis.

(3) Additional Analyses

Furthermore, binding assay was performed using ¹²⁵I-labeled Mabs (seeExample 1 (2)) to evaluate the binding affinity against FZD10 moleculeson cell surface. For radioactive analysis, 0.5 kBq (0.001 μg antibody)¹²⁵I-labeled Mabs prepared in Example 1 (2) were added to 100 μl of cellsuspension with various amounts of non-labeled identical Mabs. Afterincubation for one hour at room temperature, the cell suspension wascentrifuged at 800×g. Supernatant was removed and the radioactivity ofcell pellet was measured.

The results showed higher binding affinity of 92-13 antibody than 93-22antibody; approximately 33% of 92-13 bound to the cells andapproximately 9% of 93-22 antibody bound to the cells under the samecondition (FIG. 1 e). The amount of the bound antibody decreased asnon-labeled antibodies were added in a dose-dependent manner.

We subsequently performed binding competition analysis of 92-13 with93-22 Mabs using flow cytometry. Cell binding of the both ofAlexa488-labeled antibodies were completely blocked by high amount ofnon-labeled antibodies (FIG. 1 f, ii and iii) to each other, suggestingthat 92-13 and 93-22 Mabs are likely to recognize very similar or sameepitope of FZD10. These findings suggest that these Mabs is able tospecifically recognize FZD10 expressed on cell surface of SS cells.

Example 3 Immunohistochemistry

To evaluate the binding specificity of 92-13 and 93-22 to human tissues,we performed immunohistochemical analysis using frozen tissue sections.Tissue sections of frozen normal adult human organs (BioChain, Hayward,Calif.) were fixed with 4% paraformaldehyde at 4° C. for 15 min, andincubated with 5 μg/ml Mabs for one hour at room temperature.Subsequently, mouse ENVISION Polymer Reagent (DAKO) was added andvisualized with peroxidase substrate (3,3′-DiaminobenzidineTetrahydrochloride).

The results are shown in FIG. 2. FIG. 2 shows immunohistochemicalanalyses in SS and normal human frozen tissue sections with no antibody(a, d, g, j, and m), 92-13 (b, e, h, k, and n) and 93-22 (c, f, i, l,and o). (a-c), synovial sarcoma; (d-f), kidney; (g-i), liver, (j-l),heart; (m-o), brain. Expectedly, we observed strong immunoreactivity toFZD10 in SS specimen (FIG. 2, a, b, and c) and placenta (data notshown), but did not detect in normal kidney, heart, brain and liver(FIG. 2, d-o), as concordant with the results of northern-blot andRT-PCR experiments (Nagayama, S., et al. (2005). Oncogene, 24,6201-6212.).

Example 4 Biodistribution of Anti-FZD10 Mabs in Balb/c Mice XenograftModel

Distribution of 92-13 and 93-22 in in vivo model was examined in BALB/cmice by means of two independent methods, radionuclide imaging andfluorescent imaging.

(1) In vivo Radionuclide Imaging

In vivo experiments were performed in the animal facility in accordancewith institutional guidelines. BALB/cA Jcl-nu mice (female, 7 weeks old)were injected subcutaneously (s.c.) with SYO-1 tumor cells (5×10⁶cells), in 0.1 ml PBS, in the flanks. For biodistribution studies, micewith fully established tumors were given 10 kBq (0.5-1 μg) of¹²⁵I-labeled Mabs and 10 kBq (0.5-1 μg) of ¹¹¹In-labeled Mabs via tailvain. At 1, 24, 48 hours, animals were euthanized and the weight andradioactivity of tissues were measured. The distribution was expressedas % of injected dose/g of tissue for all samples. For optical imagingof biodistribution, LoVo-tumor bearing mice were used in addition toSYO-1 tumor mice. LoVo tumor cells (1×10⁷ cells) were injected s.c. intoBALB/cA Jcl-nu mice as described above. When tumors were fullyestablished, the mice were subjected to the imaging study.

The results in FIG. 3 a demonstrates that the radioactivity of¹¹¹In-92-13 associated with the blood decreased from 35% injected doseper gram (% ID/g) at one hour postinjection to 12% after 48 hours.Radioactivities of ¹¹¹In-92-13 associated liver, kidney, intestine,spleen, pancreas, lung, heart, stomach and muscle remained fairlyconstant or decreasing throughout the observation (FIG. 3 a).Radioactivity of ¹¹¹In-92-13 associated with tumor accumulatedthroughout the experiment, from 2% ID/g at one hour postinjection to 11%ID/g after 48 hours. On the other hand, FIG. 3 b demonstrates thatradioactivity of ¹²⁵I-labeled 92-13 associated with tumor did notincreased significantly although blood-associated radioactivity fellfrom 25% at one hour to 7% after 48 hours and radioactivities associatedwith other normal organs remained constant. The ¹²⁵I-labeled antibodieswere possibly degraded inside the cell after internalization.¹¹¹In-labeled 93-22 was also accumulated into SYO-1 tumor at 48 hourspostinjection (FIG. 3 c) and ¹²⁵I-labeled 93-22 showed poor accumulation(FIG. 3 d), suggesting its internalization as well as 92-13.

(2) In vivo Fluorescence Imaging

In vivo fluorescence imaging was performed with IVIS™ Imaging System 100series (Xenogen, Alameda, Calif.). An optimized Cy5.5 filter was used toacquire Alexa647-Mabs fluorescence in vivo. SYO-1 tumor-bearing micewere injected 20 μg of Alexa647-labeled Mabs intraperitoneally andsubjected to fluorescent imaging at various time points. The mice werefed with food that is not containing alfalfa for four days in prior toinjecting Mabs in order to reduce the background fluorescence. Whenacquiring images, mice were anesthetized with 2% of isoflurane (AbbottLaboratories) and placed in the IVIS system. The mice were euthanized atfour days after the Mab injection, the tumor and major organs weredissected, and fluorescence image was obtained.

As shown in FIG. 4 a, significant amount of fluorescence was detected atthe location of tumor at 24 hours after the injection. The tumor-boundfluorescence was observed for both Mabs, 92-13 and 93-22; the signalsreached at maximum level at about 48 hours after the injection, andcould be detectable at 96 hours after the injection. The presentinventors sacrificed these mice at 120 hours postinjection and measuredtheir fluorescence intensity in the tumor and also important normalorgans (liver, spleen, kidney, pancreas, colon) (FIGS. 4 b and 4 c).Very strong fluorescence signal was observed in the dissected tumor,whereas no fluorescence signal was detected in normal organs. Tovalidate the binding specificity, the present inventors generatedxenografts using antigen-negative cell line, LoVo, in nude mice andinjected Alexa647-labeled Mabs, performed fluorescent imaging analysis.In LoVo-bearing mice, fluorescent was detected neither at the locationof the tumor (FIG. 5 a), nor in the dissected tumor or other organs(FIGS. 5 b and 5 c). These results demonstrated that these Mabs are alsoable to bind specifically to FZD10-expressed tumor cells in vivo.

Example 5 Internalization of Anti-FZD10 Mabs into Antigen-Positive Cells

To investigate molecular behavior of these Mabs after binding to thecell surfaces, their localization was traced using in vitro imagingsystem.

Cells were plated into 8-well chamber slides (Nalge Nunc International,Naperville, Ill.) at density of 5×10⁴ cells per well. Cells wereincubated with Mabs for three hours at 37° C. in air chamber containing5% CO₂. Mabs bound to the cell surface were removed by acid strippingbuffer (0.1M Glycine, 500 mM NaCl, pH2.5) at 4° C. for 10 min andneutralized with 500 mM Tris (pH7.5). Cells were then fixed with 3.7%formaldehyde for 15 min at room temperature, and permeabilized byexposure to 0.2% TritonX-100 for 10 min, followed by blocking with 3%bovine serum albumin for one hour at room temperature. To detect theMabs internalized into the cell, samples were incubated withAlexa488-labeled goat-anti mouse IgG (1:700 dilution) for one hour atroom temperature. The slides were mounted with DAPI (Vectashield, VectorLaboratories, Burlingame, Calif.) and analyzed under Leica TCS SPIconfocal optics.

As shown in FIG. 6, both Mabs 92-13 and 93-22 were efficientlyincorporated into the cytosol of SYO-1 cells and YaFuSS cells at 3 hoursafter the incubation of Mab with cells by confocal microscope imagingdetected using Alexa488-labeled goat anti-mouse IgG (FIG. 6, a-f). Onthe other hand, the fluorescence signals of these Mabs were hardlydetectable in LoVo cells without FZD10 expression (FIG. 6, g-i),demonstrating that the specific binding of Mabs to cell-surface FZD10induced the internalization of the antibodies.

Example 6 Specific Cytotoxity of Mabs

92-13 and 93-22 had no effect on tumor cell growth when added directlyinto the cultured cell (data not shown). For therapy studies, SYO-1tumors were grown in BALB/cA Jcl-nu mice in the same manner as inExample 4. The diameters of the tumors were measured by calipers and thetumor volumes were determined using the following formula; 0.5×(largerdiameter)×(smaller diameter)² as described previously (Nagayama, S., etal. (2005). Oncogene, 24, 6201-12.). When the tumor volumes reached morethan 0.4-2.8 cm³, Balb/c-nude mice bearing subcutaneous SYO-1 tumor wererandomly assigned to treatment groups and received intravenousinjections of the 100 μCi of ⁹⁰Y-labeled Mabs or control Mabs via tailvain. Mice were weighed and tumor diameters were recorded.

FIG. 7 showed that tumor volumes were markedly reduced immediately aftertreatment, almost to traces within one week in all mice. When 50 μCi of⁹⁰Y-DTPA-92-13 were given to the mice, tumors >1 cm³ volumes refractedtwo weeks after treatment although they showed marked reduction of tumorsize immediately after treatment. The mice showed temporary decrease ofthe weight (10˜15%), however, they recovered in one week and no visibletoxic signs were observed (data not shown).

Example 7 Generation of Chimeric Antibodies

Chimeric antibodies corresponding to mouse 92-13 and 93-22 antibodies,ch92-13 and ch93-22 were generated by replacement of the variable regionsequence of each mouse antibody to the human IgG₁ constant region underthe control of CMV promoter. Total RNAs were extracted from hybridomaclones 92-13 and 93-22. cDNA was synthesized from the total RNA usingGeneRacer™ Kit (Invitrogen). The sequences of variable regions ofmonoclonal antibodies were amplified using forward primer(GeneRacer™5′Primer) and reverse primer; CH1 (IgG2a);5′-AATTTTCTTGTCCACCTTGGTG-3′ (SEQ ID NO: 3) for heavy chain and CL1(kappa); 5′-CTAACACTCATTCCTGTTGAAGCTCT-3′ (SEQ ID NO: 4) for lightchain. PCR products were sequenced and the sequences coding the m92-13and m93-22 variable region were determined.

As a result, the amino acid sequence of mouse Ig H-chain variableregions and L-chain variable regions were determined as follows:

92-13, H-Chain Variable Region:

MKCSWVIFFLMAVVTGVNSEVQLQQSGAELVKPGASVKLSCTASGFNINDTYMHWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGARGSRFAYWGQGTLVTVSA (SEQ ID NO: 13) encoded by the nucleotidesequence of SEQ ID NO: 14, and

92-13, L-Chain Variable Region:

MSVPTQVLGLLLLWLTDARCDIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYVATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHF WGTPYTFGGGTKL(SEQ ID NO: 21) encoded by the nucleotide sequence of SEQ ID NO: 22; and

93-22, H-Chain Variable Region:

MGWSRIFLFLLSITAGVHCQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGQGLEWIGRIYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARGGNYGWFAYWGQGTLVTVSAGS (SEQ ID NO: 29) encoded by the nucleotidesequence of SEQ ID NO: 30, and

93-22, L-Chain Variable Region:

METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELYTFGGGTKLGS (SEQ ID NO: 37) encoded by the nucleotide sequence ofSEQ ID NO: 38. Underlines indicate the signal sequences.

The CDR (complementarity determining region) sequences of the antibodieswere determined as follows:

92-13, INDTYMH (SEQ ID NO: 15) as VH CDR1, RIDPANGNTKYD (SEQ ID NO: 17)as VH CDR2, and GSRFAY (SEQ ID NO: 19) as VH CDR3, RASENIYSNLA (SEQ IDNO: 23) as VL CDR1, VATNLAD (SEQ ID NO: 25) as VL CDR2, and QHFWGTPY(SEQ ID NO: 27) as VL CDR3; and

93-22, SSWMN (SEQ ID NO: 31) as VH CDR1, RIYPGDGDTNYN (SEQ ID NO: 33) asVH CDR2, and GGNYGWFAY (SEQ ID NO: 35) as VH CDR3, RASKSVSTSGYSYMH (SEQID NO: 39) as VL CDR1, LASNLES (SEQ ID NO: 41) as VL CDR2, and QHSRELY(SEQ ID NO: 43) as VL CDR3.

In addition, the amino acid sequences of the H chains and L chains ofmouse monoclonal antibodies 92-13, 93-22 and 39-10 are determined asfollows:

92-13, H chain: SEQ ID NO: 58 (encoded by the nucleotide sequence of SEQID NO: 57);

92-13, L chain: SEQ ID NO: 60 (encoded by the nucleotide sequence of SEQID NO: 59);

93-22, H chain: SEQ ID NO: 62 (encoded by the nucleotide sequence of SEQID NO: 61);

93-22, L chain: SEQ ID NO: 64 (encoded by the nucleotide sequence of SEQID NO: 63);

39-10, H chain: SEQ ID NO: 66 (encoded by the nucleotide sequence of SEQID NO: 65);

39-10, L chain: SEQ ID NO: 68 (encoded by the nucleotide sequence of SEQID NO: 67).

According to the determined sequence, specific primers for m92-13variable region were designed:5′-AATAGCGGCCGCACCATGAAATGCAGCTGGGTTATCTT-3′ (SEQ ID NO: 5) and5′-AATAGCTAGCTGCAGAGACAGTGACCAGAGTCC-3′ (SEQ ID NO: 6) for heavy chainand 5′-AATAGCGGCCGCACCATGAGTGTGCCCACTCAGG-3′ (SEQ ID NO: 7) and5′-TTCCAGCTTGGTCCCCCC-3′ (SEQ ID NO: 8) for light chain. Also, specificprimers for m93-22 variable region were designed,5′-AATAGCGGCCGCACCATGGGATGGAGCCGGATCTTT-3′ (SEQ ID NO: 53) and5′-AATAGGATCCTGCAGAGACAGTGACCAGAGTCCCTT-3′ (SEQ ID NO: 54) for heavychain and 5′-AATAGCGGCCGCACCATGGAGACAGACACACTCCT-3′ (SEQ ID NO: 55) and5′-AATAGGATCCCAGCTTGGTCCCCCCTCCGAACGT-3′ (SEQ ID NO: 56) for lightchain. To construct the expression vector for chimeric antibodies, twocassette vectors were prepared. The DNA fragment coding human IgG1(CH1-CH3) was inserted into pQCXIH (Clontech) (pQCXCHIH) and the DNAfragment coding human Igκ (CL1) was inserted into pQCXIP (pQCXCLIP). Forobtaining DNA fragments coding human IgG1 or human Igκ, human constantregion library was prepared using cDNA from human PBMC (peripheral bloodmononuclear cells) by the reported method (Liu, A. Y. et al., Proc.Natl. Acad. Sci. USA, Vol. 84, 3439-43, 1987; Reff, M. E. et al., Blood,Vol. 83, No. 2, 435-45, 1994). The DNAs coding variable region of m92-13and m93-22 heavy chain and light chain were PCR amplified, sequenced andsubcloned into pQCXCHIH and pQCXCLIP respectively using NotI and BamHIsite. These vectors were co-transfected into CHO cells. Transfectedcells were cultured in F-12 medium containing 500 μg/ml Hygromycin and10 μg/ml Puromycin. When cells grow sub-confluently, the medium wasexchanged to serum-free medium (CHO-S-SFM II; GIBCO) and chimericantibody was purified from the supernatant of cultured cells usingprotein A-affinity column (GE Amersham) and was sequenced. The sequenceof heavy chain of chimeric antibody ch92-13 comprises SEQ ID NO: 46encoded by the nucleotide sequence of SEQ ID NO: 45; and the sequence oflight chain of chimeric antibody ch92-13 comprises SEQ ID NO: 48 encodedby the nucleotide sequence of SEQ ID NO: 47. The sequence of heavy chainof chimeric antibody ch93-22 comprises SEQ ID NO: 49 encoded by thenucleotide sequence of SEQ ID NO: 50; and the sequence of light chain ofchimeric antibody ch93-22 comprises SEQ ID NO: 52 encoded by thenucleotide sequence of SEQ ID NO: 51.

Example 8 Binding Activity of Chimeric Antibodies

Antibody-dependent cell cytotoxity (ADCC) activities induced by chimeric92-13 and 93-22 were determined using LDH activity as describedpreviously (Nagayama, S., et al. (2005). Oncogene, 24, 6201-6212.).Fresh effector cells were isolated from heparinized peripheral blood ofa healthy donor by Ficoll-Plaque (Amersham Bioscience). Effector cells(E) and target cells (T) (each, 5×10³/well) were co-incubated for 6 h at37° C. in quadruplicate at various E:T ratios, together with chimeric92-13, chimeric 93-22 or non-immunized human IgG, in 0.1 ml of phenolred-free RPMI 1640 supplemented with 5% FBS in a 96-well plate. LDHreleased in the culture supernatants was determined by absorbance at 490nm. The percentage of specific cytotoxicity was calculated according tothe manufacturer's instructions.

Referring to the effector activity, both chimeric 92-13 and 93-22induced ADCC specifically to the FZD10-overexpressing SYO-1 cells (FIG.8, a and c), but not to the FZD10-negative LoVo cells (FIG. 8, b and d).Particularly, chimeric 92-13 showed higher induction of cytotoxity ascompared with chimeric 93-22; however, their activity depends oneffector cell donor, possibly caused by polymorphism of Fc receptor.

Each of the applications and patents mentioned in this document, andeach document cited or referenced in each of the above applications andpatents are hereby incorporated herein by reference. Furthermore, alldocuments cited in this text, and all documents cited or referenced indocuments cited in this text, and any manufacturer's instructions orcatalogues for any products cited or mentioned in this text, are herebyincorporated herein by reference.

Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments and that manymodifications and additions thereto may be made within the scope of theinvention. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the claims. Furthermore, various combinations of the features of thefollowing dependent claims can be made with the features of theindependent claims without departing from the scope of the presentinvention.

1. An antibody or a fragment thereof, which comprises an H (heavy) chainV (variable) region comprising a complementarity determining region(CDR) having the amino acid sequences shown in SEQ ID NOs: 15, 17 and 19and an L (light) chain V region comprising a CDR having the amino acidsequences shown in SEQ ID NOs: 23, 25 and 27, and which is capable ofbinding, to a Frizzled homologue 10 (FZD10) protein or a partial peptidethereof.
 2. The antibody or fragment thereof according to claim 1,wherein the antibody is selected from the group consisting of a mouseantibody, a chimeric antibody, a humanized antibody, an antibodyfragment, and single-chain antibody.
 3. The antibody or fragment thereofaccording to claim 1, wherein the antibody is a mouse antibody.
 4. Theantibody or fragment thereof according to claim 3, wherein the mouseantibody comprises an H chain having the amino acid sequence shown inSEQ ID NO: 57 and/or an L chain having the amino acid sequence shown inSEQ ID NO:
 59. 5. The antibody or fragment thereof according to claim 3,wherein the mouse antibody is produced by the hybridoma clone 92-13(FERM BP-10628).
 6. The antibody or fragment thereof according to claim1, wherein the antibody is a chimeric antibody.
 7. The antibody orfragment thereof according to claim 6, wherein the chimeric antibodycomprises an H chain V region having the amino acid sequence shown inSEQ ID NO:
 13. 8. The antibody or fragment thereof according to claim 6,wherein the chimeric antibody comprises an H chain having the amino acidsequence shown in SEQ ID NO:
 46. 9. The antibody or fragment thereofaccording to claim 6, wherein the chimeric antibody comprises an L chainV region having the amino acid sequence shown in SEQ ID NO:
 21. 10. Theantibody or fragment thereof according to claim 6, wherein the chimericantibody comprises an L chain having the amino acid sequence shown inSEQ ID NO:
 48. 11. The antibody or fragment thereof according to claim6, wherein the chimeric antibody comprises an H chain V region havingthe amino acid sequence shown in SEQ ID NO: 13 and an L chain V regionhaving the amino acid sequence shown in SEQ ID NO:
 21. 12. The antibodyor fragment thereof according to claim 6, wherein the chimeric antibodycomprises an H chain having the amino acid sequence shown in SEQ ID NO:46 and an L chain having the amino acid sequence shown in SEQ ID NO: 48.13. The antibody or fragment thereof according to claim 6, wherein thechimeric antibody further comprises a human antibody C (constant)region.
 14. The antibody or fragment thereof according to claim 1,wherein the antibody is a humanized antibody.
 15. The antibody orfragment thereof according to claim 14, wherein the humanized antibodyfurther comprises a human antibody FR (framework) region and/or a humanantibody C region.
 16. A hybridoma clone 92-13 (FERM BP-10628) whichproduces the mouse monoclonal antibody 92-13.
 17. A pharmaceuticalcomposition, comprising the antibody or fragment according to claim 1and a pharmaceutically acceptable carrier or excipient.
 18. A kit fordiagnosis or prognosis of a disease associated with Frizzled homologue10 (FZD10), comprising the antibody or fragment according to claim 1.19. A chimeric or humanized antibody or fragment thereof which binds theFZD10 protein and in which the complementarity determining regions(CDRs) are the CDRs of the mouse monoclonal antibody 92-13 (FERMBP-10628).
 20. The antibody or fragment thereof according to claim 19,which is a chimeric antibody that further comprises a human antibody C(constant) region or which is a humanized antibody that furthercomprises a human antibody FR (framework) region and/or a human antibodyC region.
 21. A chimeric antibody or fragment thereof which comprises:(i) an H chain V region having the amino acid sequence of SEQ ID NO: 13and an L chain V region having the amino acid sequence of SEQ ID NO: 21;or (ii) an H chain having the amino acid sequence of SEQ ID NO: 46 andan L chain having the amino acid sequence of SEQ ID NO:
 48. 22. Apharmaceutical composition, comprising the antibody or fragmentaccording to claim 19 and a pharmaceutically acceptable carrier orexcipient.